Isolation and composition of novel glycosidases

ABSTRACT

Substantially pure glycosidases capable for cleaving selected glycosidic bonds have been described including glycosidases isolated from  Xanthomonas  and recombinant glycosidases. Substrate specificity of isolated enzymes have been identified for GlcNacβ1-x, Galα1-3R, Galα1-6R, Galβ1-3R, Fucα-2R, Fucα1-3R, Fucα1-4R, Manα1-2R, Manα1-3R, Manα1-6R, Manβ1-4R, Xylβ1-2R, Glcβ1-4R, and Galβ1-4R providing improved capability for selectively cleaving a glycosidic linkage in a carbohydrate substrate and for forming modified carbohydrates.

RELATED APPLICATIONS

This is a Divisional Application of U.S. application Ser. No. 08/560,809filed on 21 Nov. 1995 which is a CIP of Ser. No. 08/596,250 filed 24Jun. 1996 (U.S. Pat. No. 5,770,405 issued on 23 Jun. 1998) which is theNational Stage of PCT US94/10758 filed on 22 Sep. 1994 which is a CIP ofSer. No. 08/126,174 filed 23 Sep. 1993 (now abandoned).

TECHNICAL FIELD

The present invention relates to novel glycosidases and their uses.

BACKGROUND OF THE INVENTION

The recognition that carbohydrates play a key role in biologicalprocesses of living organisms has made their study of great importancefor medicine and basic science. The understanding of carbohydrates haslagged behind that of other types of biological molecules because of theimmense complexity and variety of these molecules and the lack ofavailability of analytic and synthetic tools that enable scientists todifferentiate one form from another.

Forms of Carbohydrates in Nature.

In nature, carbohydrates exist as polymers known as polysaccharides,that consist of a series of monosaccharides that are covalently attachedby glycosidic bonds to form both branched and linear macromolecules. Inaddition, polysaccharides or, more commonly, oligosaccharides may becoupled to macromolecules such as proteins or lipids to formglycoproteins or glycolipids. Unlike naturally occurringpolysaccharides, the oligosaccharides associated with protein or lipidconsist of a relatively small subset of monosaccharide types.

Oligosaccharides associated with glycoproteins have been the focus ofmuch of the carbohydrate research to date largely because the biologicalproperties of these molecules are diverse and their relatively shortmonosaccharide sequences make the oligosaccharides amenable to study.

Structural Features of Glycoproteins.

Glycoproteins are characterized into two groups according to theirlinkage to protein. The O-glycosyl linked oligosaccharides includingmucin-type oligosaccharides, the proteoglycan type, the collagen-typeand the extensin-type are bonded to the hydroxyl oxygen of L-serine orL-threonine. The N-glycosyl linked oligosaccharides are bound to theamido nitrogen of asparagine in a tripeptide generally of the formAsn-Xaa-Ser/Thr (where Xaa represents any amino acid). The N-linkedoligosaccharides are further differentiated into 3 subgroups these beingthe high mannose type, the complex type and the hybrid type. N-linkedoligosaccharides are frequently branched where branching commonly occurseither at a mannose residue or at an N-acetylglucosamine residue. Thesebranched structures are called biantennary, if there are two branches,and triantennary if there are three branches.

The oligosaccharide can be characterized by its sequence ofmonosaccharides. The oligosaccharide is attached at its reducing end tothe amino acid sequence of the protein while the non-reducing end isfound at the terminal monosaccharide at the other end of theoligosaccharide. Other important characteristics of oligosaccharides arethe glycosidic bonds that connect individual monosaccharides. Theglycosidic bonds obtain their numerical assignment according to thecarbons in the monosaccharide ring where linkage occurs. The carbons arenumbered in a clockwise direction from 1 to 6. Any of these carbons canbe involved in the glycosidic bond although commonly the carbon-1 on themonosaccharide closer to the non-reducing end forms a glycosidic bondwith any other carbon on the monosaccharide toward the reducing end ofthe oligosaccharide. Because each carbon on a monosaccharide isasymmetric, the glycosidic bond occurs in two anomeric configurations,the alpha and the beta anomer. The type of anomer is determined by theposition of the reactive hydroxyl group on the carbon. FIG. 1illustrates the possible linkage configurations that may exist betweentwo monosaccharides.

Synthesis and Degradation of Oligosaccharides.

Oligosaccharides are synthesized by a battery of enzymes in the cellknown as glycosidases and glycosyltransferases. Typically, anoligosaccharide is assembled on a lipid carrier and transferred to theappropriate amino acid within the protein to be glycosylated.Glycosidase trimming and glycosyltransferase mediated synthesis followsand individual monosaccharides or preassembled oligosaccharide units areremoved or added. In addition, microscopic reversibility may occur whenthe exoglycosidases that are usually hydrolytic enzymes, act astransferases in a synthetic role (Ichikawa et al. 1992, Anal. Biochem.202:215–238). In some cases, removal of a monosaccharide results in aconformational change that facilitates further chain synthesis (Camirandet al. 1992, J. Biol. Chem., 266:15120–15127). While not wishing to bebound by theory, one cause of inter-cellular variability inglycosylation patterns for a single protein may arise from differentamounts and types of available glycosidases and glycosyltransferases inany single cell.

The availability of individual glycosidases and glycosyltransferasesdepends on the nutritional environment of the cell (Goochee and Monica1990, Bio/Technology 6:67–71) the type of cell (Sheares and Robbins1986, PNAS 83:1993) and its homeostatic state (Kobata 1988, Gann Monogr.Cancer Res. 34:3–13). Associated with the variation in amounts and typeof these intracellular enzymes is the occurrence of multiple glycoformsof a single glycoprotein (Parekh et al. 1987, EMBO 6:1233–1244). Theseglycoforms differ in their oligosaccharide sequence and linkagecharacteristics as well as in the position and number of attachmentsites of the oligosaccharide to the protein. Variation in glycosylationof a single glycoprotein made in different cell types is an importantaspect of recombinant protein therapeutic production because of thepossible impact of structural heterogeneity on biological function(Sasaki et al. 1987, J. Biol. Chem. 262:12059–12076; Dube et al. 1988,J. Biol. Chem. 263:17516–17521; Lund et al. 1993, Human Antib.Hybridomas, 4:20–25; Parekh et al. 1989, Biochem. 28:7644–7662; Kagawaet al. 1988, J. of Biol. Chem. 263:17508–17515; Parekh et al. 1989,Biochem. 28:7662–7669; Parekh et al. 1989, Biochem. 28:7670–7679).

Not only does the glycosylation pattern of a single protein varyaccording to which cell it is events may be characteristic of certainevolutionarily related animal species only. Galili et al. 1987,Immunology 84:1369–1373 and Galili et al. 1988, J. Biol. Chem.263:17755–17762 identified the occurrence of Galα1-3Gal in non-primatemammals and New World monkeys, a glycosylation pattern that was absentin humans and Old World monkeys. The absence of this structure could bedemonstrated because the disaccharide elicits an immune response inhumans. The immune response to atypical glycosylation patterns presentsa yet unsolved antigenicity problem that arises from using glycoproteinsderived or manufactured in non-primate sources.

Oligosaccharides are degraded by glycosidases that are often highlyspecific for the glycosidic linkage and the stereochemistry of theoligosaccharide. An example of the influence of remotely locatedmonosaccharides on the digestion of oligosaccharides is found in humanpatients suffering from fucosidosis. These patients lack theexoglycosidase required to remove fucose from N-linked oligosaccharidesprior to digestion with endoglycosidase. The fucose interferes with theenzymatic activity of the endoglycosidase and causes undigestedoligosaccharides to be excreted in their urine. (Kobata 1984, TheBiology of Carbohydrates, Eds., Ginsberg and Robbins, Wiley, N.Y. vol.2, pp. 87–162.)

The Biological Impact of Glycosylation of Proteins.

The importance of correct synthesis and degradation of oligosaccharidesfor the organism has been demonstrated in diseases which result from asingle defective glycosidase giving rise to incorrect processing ofcarbohydrate structures. In the example cited above, disease resultsfrom the absence of a Fucosidase resulting in incorrect processing ofthe glycoprotein. Other examples include human α-Mannosidosis in whichthe major lysosomal α-Mannosidase activity is severely deficient(Gasperi et al. 1992, J. of Biol. Chem. 267:9706–9712). Aberrantoligosaccharide structures have also been associated with cancer (Sanoet al. 1992, J. Biol. Chem. 267:1522–1527).

The oligosaccharide side chains of glycoproteins have been implicated insuch cellular processes as protection of peptide chains againstproteolytic attack, facilitation of secretion to the cell surface,induction and maintenance of the protein conformation in a biologicallyactive form, clearance of glycoproteins from plasma and antigenicdeterminants in differentiation and development. In fact, at anydevelopmental stage, cells may have solved the biosynthetic problem ofcontrolled variation by making not just one glycoprotein but by codingfor large repertoires of a protein, each variant having a differentcovalently attached oligosaccharide (glycoform). The extent ofvariability that arises from multiple glycosylation sites on a peptideor indeed multiple forms of a single glycosylation site have beendiscussed by Rademacher et al. 1988, Ann. Rev. Biochem. 57:785–838, forrecombinant proteins. Because the characteristics of glycoprotein aswell as its biological properties and function vary according to thesequence and structure of the attached oligosaccharides (Cumming 1991,Glycobiology 1:115–130), the analysis of glycoprotein structure hasbecome an important requirement in characterizing recombinantpharmaceutical proteins.

New methods of analyses are required to facilitate quality control ofmanufactured pharmaceutical grade recombinant protein to permit rapid,low cost and reliable characterization of oligosaccharides todistinguish between closely related structures (Spellman 1990, Anal.Chem. 62:1714–1722). New methods to manipulate and modifyoligosaccharides on glycoproteins is desirable to improve productionlevels from cells and to optimize the biological function of proteins astherapeutic agents.

A rapid and simple method of oligosaccharide sequence and linkageanalysis would have utility in directing synthesis and analyzingfunction of glycoproteins and carbohydrates in general as well asproviding insights into the causes and implications ofmicroheterogeneity in glycosylated molecules made in differentorganisms, organs or cells as well as within a single cell.

Methods of Analyzing Carbohydrate Structures.

Existing methods for analyzing carbohydrate structure rely on complexmulti-step procedures. These procedures involve techniques such as massspectrometry, NMR, fast atom bombardment, complex chromatographytechniques (high pressure liquid chromatography, gas phasechromatography, ion-exchange and reverse-phase chromatography) andcomplex series of chemical reactions (methylation analysis, periodateoxidation and various hydrolysis reactions) and have all been used invarious combinations to determine the sequence of oligosaccharides andthe features of their glycosidic linkage. Each method can providecertain pieces of information about carbohydrate structure but each hasdisadvantages. For example, fast atom bombardment (Dell 1987, Advancesin Carbohydrate Chemistry and Biochemistry 45:19–73) can provide somesize and sequence data but does not provide information on linkagepositions or anomeric configuration. NMR is the most powerful tool foranalyzing carbohydrates (Vliegenthart et al. 1983 Advances inCarbohydrate Chemistry 41:209–375) but is relatively insensitive andrequires large quantities of analyte. These methods have been reviewedby Spellman 1990, Anal. Chem. 62:1714–1722; Lee et al. 1990, AppliedBiochem. and Biotech. 23:53–80; Geisow 1992, Bio/technology 10:277–280;Kobata 1984. Many of the above procedures require expensive equipment aswell as considerable technical expertise and technical support for theiroperation that limits their use to a few specialist laboratories.

Carbohydrate Analyses Using Glycosidases.

Enzymes have been used at various stages of carbohydrate analysis as onestep in the multi-step analyses. These enzymes include glycoamidaseshaving the ability to cleave between the glycan portion and the aminoacid (commonly Asparagine) of the protein with which it is associated.Most important are the endoglycosidases and exoglycosidases which areboth hydrolases and are so named because of their ability tospecifically cleave glycosidic bonds either within the carbohydratestructure (endo-) or at the terminal monosaccharides (exo-) at thenon-reducing end of the molecule.

Endoglycosidases have been described that cleave oligosaccharides at thereducing end at the penultimate monosaccharide to the amino acidattachment site on the peptide. Five endo-β-N-Acetylglucosaminidaseshave been purified sufficiently for use in structural studies eachhaving a different substrate specificity (Kobata 1984). In addition, anendo-α-N-acetylgalactosaminidase has also been isolated (Umemoto et al.1977, J. Biol. Chem. 252:8609–8614; Bhavanandan et al. 1976, Biochem.Biophys. Res. Commun. 70:738–745). The specificity of theseendoglycosidases make them powerful tools in analyzing oligosaccharidestructure. At this time, endoglycosidases have limited applicability dueto the small number of characterized enzymes currently commerciallyavailable. An increased number of characterized endoglycosidases havingdifferent specificities would be of utility in carbohydrate analyses.

Oligosaccharides released by endoglycosidase digestion or by chemicalmeans may be further characterized by exoglycosidase digestion.Exoglycosidases are hydrolases that cleave monosaccharide units from thenon-reducing terminus of oligosaccharides and polysaccharides. Becauseexoglycosidases have known specificities for different terminalmonosaccharides as well as for different anomeric forms, they have beenused to sequence oligosaccharides. Sequential exoglycosidase digestionused in conjunction with gel permeation chromatography was firstdescribed by Yashita et al. in 1982 (Methods in Enzymology 83:105–126).Edge et al. (1992, PNAS 89:6338–6342) described multiplex enzymereaction digestions and analysis of a sequence by analysis of arrays ofenzyme digestions. The power of sequencing oligosaccharides usingglycosidases has been limited by the availability of enzymes withwell-characterized substrate specificities. The limitations ofsubstrates for analyzing glycosidase activity has also resulted inincomplete data on glycosidic linkages between monosaccharides. As aresult, it has been necessary to conduct methylation analysis todetermine glycosidic linkages subsequent to sequence analysis.

Exoglycosidases have been isolated from diverse sources includingbacteria, viruses, plants and mammals and have specificities for sialicacid (α anomer), galactose (α and β), N-acetylglucosamine (αand β),N-acetylgalactosamine (α and β), mannose (α and β (Sano et al. 1992, J.Biol. Chem. 267:1522–1527; Moremen et al. 1991, J. Biol. Chem.266:16876–16885; Camirand et al. 1991, J. Biol. Chem. 266:15120–15127;Gasperi et al. 1992, J. Biol. Chem. 267:9706–9712; Ziegler et al. 1991,Glycobiology 1:605–614; Schatzle et al. 1992, J. Biol. Chem.267:4000–4007).

Glycosidases in the prior art have been defined in most examples bytheir substrate specificity where the characterization of the enzyme islimited by the availability of suitable substrates and the complexity ofthe assay. Furthermore, enzymes in the prior art are frequently named inan arbitrary fashion, where the names suggest biological activities thathave never been demonstrated. Limitations in the characterization ofcrude extracts or purified enzymes arise in the prior art because of thelack of suitable assays that identify what substrates are cleaved andwhat substrates are not cleaved by any single enzyme. Associated withthe problems of characterizing the enzymes are problems associated withidentifying contaminating glycosidase activity. Furthermore, not onlyare glycosidase preparations commonly contaminated with otherglycosidases they are also contaminated with proteases. The limitationsin characterizing enzymes cited in the prior art and the difficulties inobtaining substantially pure preparations of glycosidases is reflectedin the sparsity of the list of commercially available glycosidases (seeTable 1).

The substrates most commonly used in the prior art are derivatizedmonosaccharides (p-nitrophenyl-monosaccharide or 4-methylumbelliferylmonosaccharide). Whereas these substrates may provide information onsome of the monosaccharides that are recognized by glycosidases, noinformation on glycosidic bond cleavage specificities can be obtainedbecause the monosaccharide is chemically linked to the chromogenicmarker and is not linked through a glycosidic linkage to a secondmonosaccharide. In addition the derivatized substrates are of limiteduse in characterizing the recognition site of a glycosidase.Glycosidases that cleave the monosaccharide derivative, do not alwayscleave the same monosaccharide in an oligosaccharide. Likewise,glycosidases that cleave an oligosaccharide may not cleave a derivatizedsubstrate (Gasperi et al. 1992, J. Biol. Chem. 267:9706–9712).

A systematic approach is required to develop a set of labelledoligosaccharides suitable for characterizing the recognition site andthe glycosidic cleavage site of a glycosidase. In addition to providingsuitable substrates, simple rapid methods of analyzing the products of asingle or multiple glycosidase reaction are required to accomplish thescreening of a single glycosidase against multiple substrates or ofmultiple glycosidases against a single substrate.

Many of the glycosidases that are currently available have importantlimitations as analytic reagents (Jacob, et al., 1994, Methods Enzymol.230:280–299). These include the following:

1) Contamination of exoglycosidase preparations with otherexoglycosidase impurities that results in ambiguous digestion results.

2) Lack of specificity of the exoglycosidase for a specific glycosidiclinkage. Glycosidases that have been characterized appear to recognizemultiple linkages, some of these linkages being preferentiallyrecognized over others. It would be desirable to identify the extent ofpreference of any given glycosidase for a single linkage.

Furthermore, as analytic reagents, the repertoire of availableexoglycosidases of varying specificities does not provide sufficientrange to analyze and differentiate many of the linear or branchedstructures that occur in nature.

Of the available glycosidases, there is a deficit of substantially purehighly specific enzymes that have defined and reproducible substratespecificities to perform carbohydrate analyses. The deficiency in theavailability of these enzymes for carbohydrate analyses is caused atleast in part by the lack of available techniques to isolate novelglycosidases and to characterize their substrate specificities. Theavailability of a wide range of glycosidases that have definedmonosaccharide and glycosidic linkage preferences would eliminate theexisting requirement for additional types of analysis such asmethylation analysis to fully characterize an oligosaccharide and wouldprovide a powerful tool in rapid characterization of novel carbohydratestructures and their biological properties.

Source of Exoglycosidases.

A limited number of exoglycosidases are commercially available (seeTable 1). In addition, a large number of exoglycosidases have beenisolated from a variety of organisms as described above. A partial listof exoglycosidases known to be useful for sequence determinations isprovided by Linhardt et al. 1992, International Publication Number WO92/02816. An additional list of exoglycosidases is provided by Haughland1993, International Publication Number WO/93/04074. A comprehensivereview of glycosidases is provided by Conzelman et al. 1987, Advances inEnzymology 60:89; Flowers et al. 1979, Advances in Enzymology 48:29;Kobata 1979, Anal. Biochem. 100:1–14.

Although glycosidases that are presently available have been generallyisolated and manufactured from natural sources, Schatzle et al. 1992, J.Biol. Chem. 267:4000–4007, has reported cloning and sequencing thelysosomal enzyme α-Mannosidase isolated from Dictyostelium discoideum.Although Schatzle et al. characterized the structural properties of theenzyme, the substrate specificity with regard to glycosidic linkages wasnot revealed.

TABLE 1 COMMERCIALLY AVAILABLE GLYCOSIDASES LINKAGE ENZYME SOURCESPECIFICITY β-N-Acetylglucosaminidase Streptococcus pneumoniae^(OGS,BMS) 1-2,3>4,6 (+GalNAc) Chicken liver^(OGS) 1-3,4 (+GalNAc)Bovine kidney^(BMB) ? (+GalNAc) α-Fucosidase Almond meal^(G,OGS) 1-3,4Streptomyces sp 142^(T) 1-3,4 Arthrobacter^(T) 1-2 Chicken liver^(OGS)1-2,4,6 Fusarium oxysporium ^(S) 1-2,4 Bovine epididymis^(OGS)1-6>>2,3,4 Bovine kidney^(BMB) ? α-Galactosidase Coffee bean^(BMB,OGS)1-3,4,6 Mortieralla vinacea ^(S) 1-4,6 β-Galactosidase Steptococcuspneumoniae^(OGS,BMB,S) 1-4 Bovine testes^(OGS,BMB) 1-3,4>6 Jackbean^(OGS,S) 1-3,4>6 Chicken liver^(OGS) 1-3,4 α-Mannosidase Jackbean^(OGS,BMB,S) 1-2,6>3 Aspergillus saitoi^(OGS)1-2 BMB: BoehringerMannheim G: Genzyme OGS: Oxford GlycoSystems S: Seikagaku T: Takara

For the foregoing reasons, there is a need for novel substantially pureglycosidases suitable as reagents having defined substrate specificitiesand where the purified enzyme preparations are in a form that providesreproducible cleavage activity. Furthermore, there is a need for methodsof isolating and manufacturing a wide array of these enzymes suitablefor analyzing the wide variety of carbohydrate structures that occur innature. Furthermore, there is a need for rapid, low cost, simple methodsof carbohydrate analysis so as to characterize the substratespecificities of the enzymes; to provide rapid low cost methods ofsequencing carbohydrate structures; and to modify carbohydrate moietieson glycoproteins and glycolipids for purposes of altering the biologicalproperties of such molecules. The availability of a rapid, low cost,simple method of carbohydrate analysis would provide many opportunitiesto analyze the wide variety of carbohydrate structures that occur innature, to understand the functions of these molecules and to modifytheir biological properties for useful purposes by manipulating theirstructures.

SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods thatsatisfy the need for novel, substantially pure glycosidases havingidentified substrate specificities.

A preferred embodiment is a substantially pure glycosidase obtainablefrom Xanthomonas. A form of the glycosidase is a recombinant glycosidasethat is cloned by isolating DNA from a first organism, forming a genelibrary from the DNA in a second organism and identifying recombinantclones of the second organism having glycosidase activity.

An additional preferred embodiment is a substantially pure glycosidasehaving a substrate specificity for a GlcNAcβ1-X wherein the specificityof the glycosidase for GlcNAcβ1-X is 100 fold greater than forGalNAcβ1-X.

Additional embodiments of the invention are compositions comprisingsubstantially pure Galactosidases, Fucosidases or Mannosidasesobtainable from Xanthomonas.

Additional embodiments of the invention include substantially pureglycosidases having substrate specificities for Manα1-3R glycosidiclinkage, Manβ1-4R glycosidic linkage or for Xylβ1-2R glycosidic linkage.

Embodiments of the invention include a method for modifying acarbohydrate comprising selecting at least one glycosidase derived fromXanthomonas, cleaving selected glycosidic bonds between constituentmonosaccharides of the carbohydrate by means of glycosidase digestionand forming a modified carbohydrate.

A further method of the invention is one for selectively cleaving aglycosidic linkage in a carbohydrate substrate comprising selecting aglycosidase from Xanthomonas having a substrate specificity for aglycosidic linkage, permitting the glycosidase to react with thecarbohydrate substrate and cleaving the carbohydrate substrate.

A further method of the invention comprises selectively cleaving aGlcNAcβ1-X from a carbohydrate comprising selecting a glycosidase havinga substrate specificity for GlcNAcβ1-X, the substrate specificity beingat least 100 fold greater for GlcNAcβ1-X than for GalNAcβ1-X, permittingthe glycosidase to react with the carbohydrate and cleaving theGlcNAcβ1-X.

Further embodiments of the invention include cleaving Manα1-3R orManα1-6R in a carbohydrate by selecting glycosidases capable ofselectively cleaving either of these linkages with at least 100 foldpreference over Manα1-6R or Manα1-3R respectively.

These and other features, aspects and advantages of the presentinvention will become better understood with reference to the followingdescription and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects of the invention will become better understoodwith regard to the following description, appended claims andaccompanying drawings where:

FIG. 1 shows the possible glycosidic linkages that may be formed betweentwo monosaccharides.

FIG. 2 shows the results of incubating crude extracts of Xanthomonaswith oligosaccharide substrates to determine the presence of glycosidicactivity.

FIG. 3 shows a titration of α1-3,6 Galactosidase using two fold serialdilutions of purified enzyme on a substrate (109) to determine enzymeconcentration.

FIG. 4 shows the characterization of α1-2 Fucosidase (II) and α1-3,4Fucosidase (I) using substrates 120, 95, and 113.

FIG. 5 shows the characterization of β-GlcNAcase using substrates 118and 167 to demonstrate selective cleavage of linear βGlcNAc1-X overβGalNAc1-X.

FIG. 6 shows the characterization of β-GlcNAcase using linear andbranched substrates.

FIG. 7 shows the characterization of β-GlcNAcase derived fromXanthomonas compared with Hexosaminidases derived from commercialsources where the Hexosaminidases are contaminated with additionalglycosidases.

FIG. 8 shows the characterization of β1-3>>4 Galactosidase fromXanthomonas where substrate preference for Galβ1-3R linkages overGalβ1-4R linkages are demonstrated and differentiated from commercialenzymes from chicken liver and bovine testes.

FIG. 9 shows the characterization of α1-3,6 Galactosidase with ademonstration of the lack of activity of the enzyme for Galα1-4Rlinkages found in Galactosidases from other sources (coffee bean).

FIG. 10 shows the characterization of α-Mannosidases I, II and IIIactivity on linear substrates.

FIG. 11 shows the characterization of α-Mannosidases I, II and IIIactivity on branched substrates.

FIG. 12 shows the characterization of β-Glucosidase derived fromXanthomonas where substrate preference for Gluβ1-4R linkages overGluα1-4R, GlcNAcβ1-4R linkages are demonstrated.

FIG. 13 shows the results of incubating crude extracts of Xanthomonasand Bacillus with oligosaccharide substrate 300 to determine thepresence of glycosidase activity.

FIG. 14 shows the results of incubating crude extracs of X. campestriswith p-nitrophenyl glycoside substrates to determine the presence ofglycosidase activity.

FIG. 15 shows the results of incubating crude extracts of X. campestriswith oligosaccharide substrates to determine the presence of glycosidaseactivity.

FIG. 16 shows the characterization of β-xylosidase derived fromXanthomonas using substrates 300 and 264.

FIG. 17 shows the characterization of β-mannosidase derived fromXanthomonas using substrates 259 and 300.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

“Substrate specificity” of a glycosidase is defined here and in thefollowing claims as the ability of the glycosidase to recognize aspecific monosaccharide or oligosaccharide and to cleave acharacteristic glycosidic linkage positioned in a carbohydratestructure. “A glycosidase” is defined here and in the claims as anenzyme that can catalyze the hydrolysis of the glycosidic linkagebetween two adjacent monosaccharides (wherein the monosaccharides occurwithin oligosaccharides, polysaccharides or in complex carbohydratessuch as glycoproteins and glycolipids).

“Carbohydrate” is defined here and in the claims to denoteoligosaccharides, polysaccharides or complex structures, these moleculeseither occurring freely or attached to a second molecule such as aprotein or lipid.

“Oligosaccharide” is defined here and in the claims as a series oflinked monosaccharides having a chain length in the range of two or moremonosaccharides to approximately 30 monosaccharides.

“1-X” is defined here and in the claims as a linkage between the carbon1 of a specified monosaccharide and an unspecified carbon on an adjacentunspecified monosaccharide.

“1-3R” is defined here and in the claims as a linkage between a carbon 1on a specified monosaccharide and a carbon 3 of an adjacent unspecifiedmonosaccharide (the unspecified monosaccharide “R” occurring within anoligosaccharide). Other linkages to carbon atoms other than to carbon 3can be used as long as they are specified.

“A preparation from an organism” is defined here and in the claims asincluding cell extract or media.

Abbreviations have been used as follows: Glc is glucose, Gal isgalactose, Fru is fructose, Man is mannose, GlcNAc isN-acetylglucosamine, GalNAc is N-acetylgalactosamine, Xyl is xylose, Fucis fucose, β-GlcNAcase is β-N-Acetylglucosaminidase, β-GalNAcase isβ-N-Acetylgalactosaminidase, β-Glcase is β-Glucosidase, and Co iscoumarin, AMC is 7-amino methylcoumarin, TLC is thin layerchromatography.

Development of an Assay for Glycosidic Activity

A preferred embodiment of the invention includes a method of rapidly,simply and accurately determining the digestion products of aglycosidase reaction. A suitable marker was selected to label substratesand to measure glycosidic activity. The reaction products were detectedusing a rapid and reproducible separation technology. The assay methodwas found to be sufficiently sensitive to allow detection ofcontaminating enzyme activity (FIG. 7), for determining enzyme titers byserial dilution (FIG. 3) and for determining relative affinities of asingle enzyme for single and multiple glycosidic linkages (Example 4,FIGS. 2, 4–11).

Labelled Substrates Suitable For Screening For Glycosidase Activity.

Several approaches exist for labeling substrates to determine glycosideactivity. These include:

a) Chromogenic monosaccharide derivatives. Existing methods of screeningfor glycosidases most commonly use chromogenic derivatives ofmonosaccharides, e.g. p-nitrophenylglycosides (Advanced Enzymology60:89; Tronsmo et al. 1993, Anal. Biochem. 203:74–79). Whereas cleavageof chromogenic monosaccharides provides information about thespecificity of a glycosidase for a specific monosaccharide, thesesubstrates provide little information about the specificity of theenzyme for glycosidic linkages. Furthermore, cleavage of the derivatizedmonosaccharides does not provide any information on how enzyme activityis affected by adjacent monosaccharides or other molecular structures.As a result, some glycosidases that are active on synthetic substratesare inactive on oligosaccharides substrates and vice versa (Montreuil etal., Carbohydrate Analysis: A Practical Approach, Chaplin et al. Eds.,ch. 5, pg. 143). In embodiments of this invention, p-nitrophenylsubstrates have been used for determining the specificity of newlyisolated glycosidases for selected monosaccharides.

b) Fluorescently labelled oligosaccharides. Methods are available forlabelling oligosaccharides with fluorescent amines by reductiveamination. Examples of such fluorescent amines include:7-amino-methylcoumarin (AMC) (Prahash et al. 1983, Anal. Biochem.128:41–46), 2-aminopyridine (Reinhold et al. 1983, J. Carbohydr. Chem2(1):1–18); p-aminoacetophenone, p-aminobenzoic ethyl ester and aniline(Wang et al. 1984, Anal. Biochem. 141:360–361) and others incorporatedby reference to Klock (1993) International Patent Application WO93/05076, Haugland (1993) International Patent Application (WO 93/04077)and WO 93/04074.

c) Radioactively labelled oligosaccharides. Oligosaccharides have beenstoichiometrically radiolabeled on the reducing end with NaB³H₄ forvarious analytical methods used in the structure determination ofcomplex oligosaccharides (Young et al. 1971, Biochemistry 10:3457; Tyrco1981. Anal. Biochem. 118:278–283; Wells et al. 1981, Anal. Biochem.110:397–406). Alternative methods use tritium-labeled oligosaccharides(Yamashita et al. 1980, J. Biol. Chem. 255(12):5635–5642; Fukuda 1985,Biochemistry 24:2154–2163).

In a preferred embodiment of the invention, the fluorescent chromophore,7-aminocoumarin (AMC) was selected for labelling oligosaccharidesubstrates at the reducing end. Advantages of AMC labelling include thefollowing: high quantum efficiency and excellent photostability; littleor no inhibitory effect on enzymatic cleavage of glycosidic linkagesthat are more than 1 monosaccharide removed from the reducing end; andready detectability of the chromophore labelled oligosaccharide on athin layer chromatogram.

Analysis of the Reaction Products of Glycosidases.

Once the labelled substrate(s) have been reacted with the glycosidase,the reaction products if any were characterized using a suitableseparation method. Methods for separating oligosaccharides andmonosaccharides include polyacrylamide gel electrophoresis, paperelectrophoresis, descending paper chromatography, capillaryelectrophoresis, TLC and HPLC. The characteristics of the labelledsubstrate determines in part the choice of separation technology. Forexample, Jackson (Biochem. J. 1990, 270:705–713) has described a methodrequiring the covalent labeling of oligosaccharides and smallpolysaccharides at their reducing ends using the fluorophore8-aminonapthalene-1,3,6 trisulphonic acid (ANTS). This fluorophore waspreferred for separation by polyacrylamide gel electrophoresis becausethe ionic charge imparted by the label facilitated separation of theoligosaccharide in an electric field. Indeed this method was able toresolve molecules varying in size from single monosaccharides topolymers of 26 residues on a single gel using a small amount ofmaterial. Unfortunately, the requirement for labelling the substratewith a relatively large charged marker at the reducing end can interferewith the exoglycosidase reactions. Linhardt (International publicationNo WO 92/02816) described a similar approach to that of Jackson forsequencing oligosaccharides. This approach required the addition offluorescent negatively charged groups to the reducing end of theoligosaccharide component after release from a glycoconjugate and thesubsequent separation of different oligosaccharides on polyacrylamidegels by capillary dynamic sieving electrophoresis. Modifications of thisapproach were recently reported by O'Neil (AAAS meeting in August 1993)and Higgins (AAAS meeting August 1993). A disadvantage of the aboveapproach includes the high cost of the procedure.

An alternative to polyacrylamide gel electrophoresis is that of lowpressure permeation chromatography. This approach was used by Edge etal. 1992, PNAS 89:6338–6342 to sequence oligosaccharides using a reagentarray analysis method. Edge et al. mixed radioactive oligosaccharideswith mixtures of exoglycosidases in different combinations and thenanalyzed the digestion products by Bio-Gel P4 column chromatography.

In a preferred embodiment of the invention, thin-layer silica gelchromatography (TLC) has been selected as a rapid method of separatinghydrolysis products of a glycosidase reaction, by their molecular weightand the number of hydroxyl groups and is capable of separating7-aminomethyl coumarin labelled oligosaccharides of different lengthsthat can be readily detected under UV light (see Example 2).

The sensitivity of the above technique has been demonstrated in examplesthat reveal the existence of contaminating enzymes in commerciallyavailable substrates (FIG. 7) and in examples that show that a singleglycosidase may have increased affinity for one glycosidic linkage overanother (FIG. 8).

An important advantage of TLC for the methods of the invention is thatlarge numbers of samples (using small amounts of substrate) can berapidly screened for glycosidase activity without a large investment intime and equipment (see FIG. 2). As many as 25–30 samples may be loadedonto a single standard sized silica gel TLC plate and analyzed in onebatch. To optimize the separation of AMC-labeled oligosaccharidesranging in size from 1 to 30 carbohydrate residues, polar solventsystems have been formulated for the optimization of the separation ofunmodified oligosaccharides according to the invention (Table 2).

TABLE 2 POLAR SOLVENT FORMULATIONS SUITABLE FOR THE SEPARATION OFOLIGOSACCHARIDES OF DIFFERENT SIZES ISOPROPANOL: OLIGOSACCHARIDESSOLVENT LABEL ETHANOL H₂O V:V:V: RESOLVED A 2.5:1.0:0.5 1–6 B2.0:1.0:1.0  7–10 C 1.8:1.0:1.2 10–15 D 1.4:1.0:1.6 15–20

The ability to screen large numbers of oligosaccharides for glycosidiccleavage rapidly, simply and accurately, using TLC, that forms a methodof the invention provides a novel approach to the automation ofcarbohydrate sequencing.

Automation utilizing the analysis of coumarin labelled substrates on TLCmight include the following features: forming a primary array comprisingaliquoted labelled substrates mixed with defined mixtures ofexoglycosidases. The array would be chosen to determine the type ofoligosaccharide (such as high mannose, complex or hybrid) and todetermine the general structure of the oligosaccharide in terms ofregions of mannose, galactose etc using glycosidases that are notlinkage specific. Samples from the reaction mixtures would be spottedonto TLC plates and run in various solvents (depending on the size ofthe oligosaccharide). The TLC plates would then be placed in organizedgrids to allow the fluorescence to be detected by UV light, digitizedand recorded. Markings off the grid could be compared to patterns storedin the computerized database and all possible theoretical sequencesdetermined. Ambiguities in sequence structure would then be resolved ina second round of glycosidase reactions utilizing exoglycosidases thathave substrate specificity for selected glycosidic linkages and branchedor linear molecules.

This type of approach to automated analysis has many advantages. Theseinclude a single step sequencing method, utilization of a wide range ofcharacterized glycosidases and substrates and a requirement for smallamounts of substrate.

The choice of AMC as the fluorescent marker and TLC as a separationmethodology does not however preclude other markers or other separationtechniques from being used to assay glycosidic activity according to themethods of the invention.

Screening and Characterization of Glycosidases.

A specific embodiment of the invention includes a method the screeningand for characterizing glycosidases. Organisms are selected that have anincreased probability of producing a range of glycosidases. The methodinvolves the analysis of the glycosidase hydrolysis products from crudepreparations of the organism using labelled oligosaccharide substratesor derivatized monosaccharides that have a defined length, composition,and secondary structure. Subsequently, glycosidases are isolated andfurther characterized and their substrate specificities are furtherdefined.

Screening Organisms For Novel Glycosidases.

An embodiment of the invention is the recognition that organisms thatselectively utilize carbohydrates as a food source represent a source ofnovel glycosidases. This feature is exemplified in Tables 3 and 4. Inthese Tables, cell extracts of different strains of Xanthomonas andBacillus were screened using a set of coumarin-labelled oligosaccharidesas substrates as described in Example 2. Reaction products wereidentified by TLC. In addition to cell extracts, media collected frompreparations of cells may also be screened for glycosidase activity.

A number of novel exoglycosidases were identified from Xanthomonasextracts following thin layer chromatography of reacted substrates (FIG.2). All Xanthomonas strains tested with a set of substrates had at least1 glycosidase activity. Six of seven (86%) had at least 3 glycosidaseactivities including N-Acetylglucosaminidase, Fucosidase, Galactosidaseand Mannosidase activity. In contrast, when soil derived Bacillusstrains were tested for glycosidase activity, only 2 of the 9 strainshad at least a single glycosidase activity.

With reference to Tables 3 and 4, a random screening method has beendeveloped which allows for a wide range of cell extracts, media or otherpreparations from related organisms or unrelated organisms to besystematically screened for glycosidase activity against any of a set offluorescently labelled oligosaccharides of known structure and sequence.

This novel approach revealed multiple enzyme activities in differentstrains of related organisms (Xanthomonas) (FIG. 2) which have beenfurther characterized in a single strain of organism, an example beingXanthomonas manihotis. The invention is not limited in scope toXanthomonas which serves here as an example of the utility of theinvention. Instead, the invention is applicable to a wide range oforganisms and cells.

Production of Glycosidases

The glycosidases identified by the random screening method of theinvention and subsequently isolated, purified and further screenedagainst selected substrates, may be further characterized by proteinsequencing providing a partial or complete protein sequence and a DNAcoding sequence for purposes of preparing recombinant forms of theenzyme. In an embodiment of the invention, a method is described forcloning glycosidases and for screening recombinant clones so as toidentify and isolate clones (Example 5). The efficiency of isolatingrecombinant clones can be further improved by growing the recombinantlibrary on specific food sources accessible only to those organismsexpressing a specific glycosidases. An embodiment of such a screeningsubstrate includes a disaccharide (see Example 5) or an oligosaccharidelinked to pantothenic acid. The availability of cloned glycosidaseshaving a known DNA sequence further permits the genetic engineering ofthese DNA sequences to form mutant enzymes having altered substratespecificities.

Characterization of Glycosidase Activity

Subsequent to the identification of enzyme activity in a crude extract,the invention provides for the isolation and purification of theglycosidases by techniques known in the art and described more fully inExample 3 (for glycosidases derived from Xanthomonas). Following theisolation and purification of glycosidases, further characterization ofthe enzyme by its substrate specificity was performed (FIGS. 4–11).Cofactor determination and the optimal pH of the reaction was also asidentified as described in Table 5 and Example 4.

The invention is by no means restricted in scope to the substrates orthe enzymes described below. Indeed, novel enzymes resulting from thescreening method described, provide the means to construct novellabelled oligosaccharide substrates which may be further used to analyzecrude extracts of organism or cells in an iterative process.

Novel glycosidases isolated and characterized from Xanthomonas accordingto this invention have been characterized by the following features.

(a) Selective substrate specificity for different monosaccharides.Glycosidases of the invention are capable of differentiating betweenstereoisomers of pyranose monosaccharides. In particular, theβ-N-Acetylglucosaminidase of the invention has a selective affinity ofat least 100 fold for β-N-Acetylglucosamine (GlcNAcβ1-X) overβ-N-Acetylgalactosamine (GalNAcβ1-X). This is in contrast toβ-N-Acetylglucosaminidases of the prior art that do not readilydifferentiate between the two forms (FIG. 7).

(b) Ability to distinguish between anomeric forms of a singlemonosaccharide. Within the set of substrates assayed, a glycosidase hasspecificity for one anomeric form only (α or β) of a monosaccharide.

(c) Substrate specificity for selected glycosidic linkages. Theglycosidases of the invention have demonstrated selective specificityfor the following: a single glycosidic linkage (for example Fucα1-2R,Manα1-6R from Xanthomonas), or for more than one glycosidic linkage (forexample, Manα1-3R and Manα1-6R, or for Galα1-3R and Galα1-6R or Fucα1-3Rand Fucα1-4R from Xanthomonas) (FIGS. 4, 9-11; Tables 6, 8).

In some cases, a selective preference is identified for a singlelinkage. For some enzymes, cleavage of a plurality of linkages wasobserved with an established preference for one linkage over a secondlinkage (for example, β-Galactosidase obtained from Xanthomonas has aquantified preference for Galβ1-3R over Galβ1-4R (Galβ1-3>>4R) (FIG. 8).In addition, some of the glycosidases of the invention have a preferencefor cleaving substrates in a linear array whereas other glycosidases arecapable of cleaving at branch points in an oligosaccharide (α1-3,4Fucosidase, α1-3,6 Mannosidase). Whereas the glycosidases of theinvention provide reproducible cleavage profiles using availableoligosaccharide motifs, variations in cleavage patterns may arise if thesubstrate is associated with carbohydrate structures (monosaccharides,oligosaccharide or polysaccharides) or with proteins, lipids orsynthetic markers that sterically affect enzyme activity.

Although it is not possible to screen a single novel glycosidase againstall possible substrate variants, selected substrates that representcommonly occurring carbohydrate motifs have been used here tocharacterize glycosidases of the invention. The analysis however doesnot exclude the possibility that a glycosidase of the invention iscapable of recognizing an additional substrate not included in thescreening assay. Alternatively, a glycosidase may fail to recognize aknown substrate included in a moiety of a larger molecule because ofsteric effects resulting from distantly located molecules in the samestructure.

Included in the glycosidases described herein, is an α1-2 Fucosidasewith an ability to cleave Fucα1-2R linkages at a branch point. Cleavageof branched Fucα1-2R has utility in reducing the immunogenicity ofstored blood and the availability of an α-Fucosidase that selectivelycleaves this substrate, provides an approach to modifying the ABOreactivity of blood stored in blood banks. Among the glycosidasesdescribed above, the α1-3,6 Galactosidase has clinical importancebecause of its ability to cleave the antigenic Galα1-3R linkage that iscommonly terminally positioned on recombinant glycoproteins manufacturedin non-human cell lines. The removal of the Galα1-3R linkage wouldeliminate an undesirable immune response to recombinant therapeuticproteins.

The identification of α1-3,6 Mannosidase, α1-2,3 Mannosidase, the α1-3Mannosidase and α1-6 Mannosidase provide for the first time the abilityto identify and sequence the antennary branches attached to specificmannose linkages in high mannose and hybrid structures thereby providingsignificantly greater resolution of structure than previously possibleby enzymatic methods.

Applications of Glycosidases

In an embodiment of the invention, the combination of large numbers ofisolated, substantially pure glycosidases having an identified substratespecificity together with a rapid and simple assay for identifyingreaction products, provide an improved method for accomplishing thefollowing applications:

a) sequencing carbohydrate structures that occur either freely in natureor have been cleaved from proteins or lipids;

b) modifying oligosaccharides on glycoprotein, glycolipid orcarbohydrate molecules that occur freely in nature for purposes ofidentifying the biological role of the oligosaccharides or for alteringthe biological characteristics of the molecule, where the moleculesinclude therapeutic proteins;

c) purifying a desired glycosidase by column chromatography or othermeans that require analysis of fractions having glycolytic activity andallowing the detection of undesirable contaminating glycosidases;

d) manufacturing processes that require degradation of naturallyoccurring carbohydrate structures such as cellulose from plant materialfor use in the paper industry;

e) characterizing carbohydrate receptors on cells having a specificityfor selected oligosaccharide ligands;

f) investigating mechanisms of action for biological systems that relyon characteristic carbohydrate structures as described by Varki 1993,Glycobiology 3:97–130 where the cited applications are incorporated byreference.

To more easily perform the above methods, kits may be prepared whereinthe kits include a set of glycosidic enzymes isolated from naturalsources or by recombinant means (the recombinant form being manufacturedby fermentation of transformed microorganisms or from transgenic animalsand plants) being substantially pure and having identified substratespecificities suitable for sequencing carbohydrates. Such kits mayinclude reagents either singly or together that are suitable forcleaving oligosaccharides from proteins, lipids or carbohydrates andadding a fluorescent label (coumarin) at the reducing end.

Additionally, kits may be prepared wherein the kits include a set ofglycosidic enzymes isolated from natural sources or by recombinant meansbeing substantially pure and having identified substrate specificitiessuitable for identifying the biological role of carbohydrate moieties orfor altering the biological characteristics of the macromoleculeincluding therapeutic proteins.

Additionally, kits may be prepared wherein the kits include sets offluorescent labelled substrates such as coumarin labelled substratessuitable for rapidly assaying glycosidase activity during thepurification of such enzymes by column chromatography or other meansthat require analysis of fractions having glycolytic activity.

Kits may be prepared wherein the kits include enzymes suited forindustrial scale treatment of naturally occurring or syntheticcarbohydrate structures.

EXAMPLES Example 1 Preparation of Substrates for Enzyme Assays:AMC-labeling of Oligosaccharides

0.25 to 1 mg oligosaccharide (either commercially obtained from AccurateChemical and Scientific Corp., Westbury, N.Y.; Sigma Chemical, St.Louis, Mo.; Pfanstiehl Labs, Waukegan, IL.; and V-Labs Inc., Covington,La. or isolated according the method incorporated by reference fromCarbohydrate Analysis: A Practical Approach (1986) Eds. Chaplin, M. F.Kennedy, J. F. (IRL Press Limited, England) pp. 150–151)

0.1 to 5.0 μmoles oligosaccharide was dissolved in 100 μl H₂O. Theaqueous carbohydrate solution was added to a solution containing 300 μlmethanol, 20 mg (0.11 μmole) AMC(Eastman Kodak-Rochester N.Y.), 35 mg(0.55 μmole) NaCNBH₃ and 41 μl glacial acetic acid. The mixture wassealed into a screw cap microfuge tube and heated in a dry block at 80°C. for 45 minutes. The reaction was loaded onto a G-25 column (2×50 cm)equilibrated with deionized water. The product was eluted with deionizedwater and 1 ml fractions were collected. Fractions were assayed forpurity by carefully spotting (to form a band), 5 μl onto a silica gel 60TLC plate. The plate was developed by TLC as described in Example 2. Theappropriate fractions were pooled and concentrated by vacuum toapproximately 0.1–1 μmole/ml. Stock solutions were stored at −20° C.

Example 2 Method For Screening Organisms For Glycosidase Activity

Preparation of Cell Extracts For Screening Assay.

0.1–0.5 g of cell paste was thawed and suspended in three volumes ofBuffer A″ (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA). The cellsuspension was briefly sonicated before being centrifuged at 14,000 rpmfor 10 minutes at 4° C. in an Eppendorf microcentrifuge.

Glycosidase Digestion Reaction.

1–5 μl of bacterial cell extract or cell growth media or partiallypurified extracts were added to a 10 μl reaction mixture containing 1nanomole of AMC labelled substrate in 50 mM Na citrate buffer (variouspH's and cofactors; see Table 5). The reaction was incubated at 37° C.for a period in the range of 5 minutes to 20 hours. 2–3 μl of reactionwas spotted in a band onto a silica gel TLC plate as described below.One unit of enzyme was defined as the amount of enzyme required torelease 1 nmole of terminal monosaccharide from an oligosaccharidesubstrate at 37° C. in 1 hour.

Analysis of Digestion Products by Thin Layer Chromatography ofAMC-Labeled Oligosaccharides.

2–3 μl (=0.25 nmoles substrate) of glycosidase digestion reaction werespotted in a tight band (0.5 cm wide lane) onto silica gel 60 TLCglass-backed plates (0.25 mm thick, 20×20 cm). The bands were completelydried with a hot air gun (temperature should not exceed 70° C.). The TLCplate was developed until the solvent front moved 10 cm, in variousisopropanol: ethanol: H₂O mixtures (Table 1) depending on theoligosaccharide sizes. The bands were visualized with a hand-held 314 nmultraviolet lamp. A minimum of 0.1 nmol of digestion product could bedetected using this technique.

Controls included a marker consisting of an undigested disaccharide(92b) (Galβ1-4GlcNAc-Co) a tetrasaccharide (167)(Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Co) and a hexasaccharide (197)Galβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3)Galβ1Undigested substrate alsoserved as a control.

The results of screening 16 cell extracts from different bacterialstrains of Xanthomonas and Bacillus are summarized in Tables 3 and 4.All extracts of Xanthomonas cleaved at least one of 14 substrates testedwith some cleaving as many as 10 substrates indicating multiple enzymeactivities.

FIG. 2 shows the results of an analysis of seven crude extracts derivedfrom Xanthomonas strains and tested against substrate 113(Galβ1-3(Fucα1-4) GlcNAcβ1-3Galβ1-4Glc-Co) and substrate 167(Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Co).

TABLE 3 RESULTS FROM GLYCOSIDASE SCREEN BACILLUS SUBSTRATE A B C D EFGHI GlcNAcβ1-4GlcNAcβ1-4GlcNAc − − − − − −−−−GlcNAcβ1-3Galα1-4Galβ1-4Glc − − − − − −−−− Fucα1-2Galβ1-4Glc − − − − −−−−−

− − − − − −−−−

− − − − − −−−− Galα1-3Galβ1-3GlcNAc − − + − − −−−+ Galα1-4Galβ1-4Gal − −− − − −−−− Galα1-6Glcα1-2Fru − − − − − −−−− Galβ1-3GlcNAcβ1-3Galβ1-4Glc− − − − − −−−+ Galβ1-4GlcNAcβ1-3Galβ1-4Glc − − − − − −−−+Glcβ1-4Glcβ1-4Glc − − − − − −−−− Manα1-2Manα1-3Manβ1-4GlcNAc − − − − −−−−− Manα1-3Manβ1-4GlcNAc − − − − − −−−−

− − − − − −−−− A: Bacillus globigii I B: Bacillus globigii II C:Bacillus caldolyticus D: Bacillus brevis E: Bacillus stearothemophilusstrain A F: Bacillus stearothemophilus strain B G: Bacillusaneurinolyticus H: Bacillus sphaericus I: Bacillus stearothemophilusstrain C Note: Only 2 of 9 Bacillus strains tested had at least 1glycosidase activity. None had at least 3 glycosidase activities.

TABLE 4 RESULTS FROM GLYCOSIDASE SCREEN XANTHOMONAS SUBSTRATE A B C D EFG GlcNAcβ1-4GlcNAcβ1-4GlcNAc + + + + − −− GlcNAcβ1-3Galα1-4Galβ1-4Glc −− − − − −− Fucα1-2Galβ1-4Glc + − + + + +−

+ + + + + −−

+ + + + + −− Galα1-3Galβ1-3GlcNAc − − + + − −− Galα1-4Galβ1-4Gal − − − −− −− Galα1-6Glcα1-2Fru − − + + − −−Galβ1-3GlcNAcβ1-3Galβ1-4Glc + + + + + −− Galβ1-4GlcNAcβ1-3Galβ1-4Glc − −− − + −− Glcβ1-4Glcβ1-4Glc + + + + + ++ Manα1-2Manα1-3Manβ1-4GlcNAc − −− + + +− Manα1-3Manβ1-4GlcNAc + − + − + −−

+ − + − + −− A: Xanthomonas holcicola ATCC# 13461 B: Xanthomonas badriiATCC# 11672 C: Xanthomonas manihotis ATCC# 49764 D: Xanthomonascyanopsidis ATCC# 55472 E: Xanthomonas oryzae ATCC# 55470 F: Xanthomonascampestris ATCC# 55471 G: Xanthomonas campestris Note: All Xanthomonasstrains tested had at least 1 glycosidase activity. 6 of 7 (86%) had atleast 3 glycosidase activities.

Example 3 Method For Purification of Glycosidases From Xanthomonasmanihotis

Fermentation of Xanthomonas manihotis.

Xanthomonas manihotis strain NEB 257 (ATCC #49764) was grown in mediaconsisting of 1 g/l yeast extract, 2 g/l tryptone, 6 g/l sodiumphosphate (dibasic), 3 g/l potassium phosphate (monobasic), 0.5 g/lNaCl, 1 g/l ammonium chloride, 2 g/l glucose, 1 mM calcium chloride, 1mM magnesium sulfate. The cells were incubated at 30° C. until latelogarithmic stage with aeration and agitation. The cells were harvestedby centrifugation and stored frozen at −70° C.

Preparation of Crude Extract.

All further procedures were performed either on ice or at 4° C. 254grams of cell paste obtained above were suspended in two volumes ofBuffer A (20 mM Tris-HCl (pH 7.5) 50 mM NaCl, 0.1 mM EDTA). The cellsuspension was passed through a Gaulin homogenizer (Model M-15) twice at12,000 psi. The lysate was centrifuged at 1,300 g for 40 min in aSharples continuous centrifuge. 500 ml of supernatant was obtained.

Purification of Glycosidases.

Glycosidases were separated and purified from crude cell extracts byutilizing a series of separation methods that differentiated the enzymesaccording to their hydrophobicity and their charge. Enzymes were assayedaccording to the methods described in Example 2 using conditionsdescribed in Table 5.

The crude extract (500 ml) was loaded onto a column of DEAE SepharoseCL-6B (5.0×25 cm) equilibrated with Buffer A (20 mM Tris-HCl pH 7.5, 50mM NaCl, 0.1 mM EDTA). The column was washed with 2000 ml of Buffer Afollowed by a linear gradient of NaCl formed with 2000 ml of Buffer Aand 2000 ml of Buffer A containing 1 M NaCl. Fractions (21 ml) werecollected at a flow- rate of 3 ml/min. Fractions were assayed for α1-2Fucosidase activity as described above. The peak of enzyme activityeluted from the column between 0.35–0.5 M NaCl. Fractions containingα1-2 Fucosidase activity were pooled and the enzyme was further purifiedas described below in Section A. The flow through from the DEAESepharose column was collected and assayed for all other glycosidaseactivities as described above. After determining that all glycosidaseswere present, the DEAE flow through was immediately applied to a columnof Heparin Sepharose CL-6B (2.6×35 cm) equilibrated with Buffer A. Thecolumn was washed with 400 ml of Buffer A followed by a linear Gradientof NaCl formed with 500 ml of Buffer A and 500 ml of Buffer A containing0.95 M NaCl. Fractions (12 ml) were collected at a flow rate of 2ml/min. Fractions were assayed for β-GlcNAcase, α1-6 Mannosidase andα1-3,6 Galactosidase activity as described above. The peaks ofβ-GlcNAcase and α1-6 Mannosidase co-eluted from the column between0.3–0.45 M NaCl. Fractions containing both activities were pooled (130ml) and the enzymes were further purified as described below in sectionsB and C. The peak of α1-3,6 Galactosidase activity eluted from theHeparin Sepharose column between 0.45–0.55 M NaCl. Fractions containingα1-3,6 Galactosidase activity were pooled and the enzyme was furtherpurified as described below in section D. The column flow through andwash (350 ml) from the Heparin Sepharose column was collected andassayed for β1-3>>4 Galactosidase, α1-2,3 Mannosidase, β-Glucosidase andα1-3,4 Fucosidase activities as described above. All the enzymeactivities were found in the column wash. 46.25 g of ammonium sulfatewas added to the column wash with gentle stirring to a finalconcentration of 1 M. The wash was then applied to a column of PhenylSepharose (1.6×15 cm) equilibrated with Buffer B (20 mM Tris-HCl pH 7.5,1 M (NH₄)₂SO₄, 0.1 mM EDTA). The column was washed with 60 ml of BufferB followed by a linear decreasing gradient of ammonium sulfate formedwith 120 ml of Buffer B and 120 ml of Buffer B containing only 0.001 M(NH₄)₂SO₄. Fractions (4 ml) were collected at a flow rate of 2 ml/min.Fractions were assayed for β1-3>>4 Galactosidase, α1-2,3 Mannosidase andβ-Glucosidase activities described above. The peaks of β1-3>>4Galactosidase and α1-2,3 Mannosidase activities co-eluted from thecolumn between 0.6–0.35 M (NH₄)₂SO₄. Fractions containing bothactivities were pooled and the enzymes further purified as described insections E and F. The peak of β-Glucosidase activity eluted from thecolumn between 0.25–0.001 M (NH₄)₂SO₄. Fractions containingβ-Glucosidase activity were pooled and the enzyme further purified asdescribed in Section G. The column flow through and wash from the PhenylSepharose column were collected and assayed for α1-3,4 Fucosidaseactivity as described above. The wash was found to contain the peak ofα1-3,4 Fucosidase activity. Further purification of this enzyme wasperformed as described below in Section H.

A. α1-2 Fucosidase. To the DEAE Sepharose pool (300 ml) described abovethat contains α1-2 Fucosidase activity 40 g of ammonium sulfate wasadded with gentle stirring to a final concentration of 1 M. The pool wasthen applied to a column of Phenyl Sepharose (1.6×15 cm) equilibratedwith Buffer B. The column was washed with 60 ml of Buffer B followed bya linear decreasing gradient of ammonium sulfate formed with 120 mlBuffer B and 120 ml of Buffer B containing only 0.001 M (NH₄)₂SO₄.Fractions (4 ml) were collected at a flow rate of 2 ml/min. Fractionswere assayed for α1-2 Fucosidase activity as described above. The peakof enzyme activity was pooled and eluted at 0.001 M (NH₄)₂SO₄. Afterdialysis against Buffer C (20 mM Sodium acetate pH 5.2, 0.1 mM EDTA)overnight, the pooled enzyme was loaded onto a column of S-Sepharose(1.0×10 cm) equilibrated with Buffer C. The column was washed with 20 mlof Buffer C. The column flow through and wash were collected and assayedfor enzyme activity as described above. The peak of enzyme activity wasdetermined in the column wash. After dialysis against Buffer Aovernight, the wash was applied to a Mono Q HR 5/5 (1 ml) columnequilibrated with Buffer A. The column was washed with 2 ml of Buffer Afollowed by a linear gradient of NaCl formed with 40 ml of Buffer A and40 ml of Buffer A containing 0.6 M NaCl. Fractions (1.5 ml) werecollected at a flow rate of 1 ml/min and assayed for α1-2 Fucosidaseactivity. The peak of enzyme activity was pooled and eluted from thecolumn between 0.05–0.15 M NaCl. After dialysis overnight against BufferA, sodium azide was added to 0.02% before storing the enzyme at 4° C. Ayield of 1500 units of substantially pure enzyme was obtained afterpurification of the crude extract.

B. β-N-Acetylglucosaminidase. The enzyme pool (130 ml) described abovethat contains both β-N-Acetylglucosaminidase and α1-6 Mannosidaseactivities was dialyzed overnight against Buffer A. After dialysis, theenzyme pool was applied to a column of Q-Sepharose (1.6×15 cm)equilibrated with Buffer A. The column was washed with 60 ml of Buffer Afollowed by a linear gradient of NaCl formed with 120 ml of Buffer A and120 ml of Buffer A containing 1 M NaCl. Fractions (4 ml) were collectedat a flow rate of 2 ml/min. The column flow through was collected andassayed for β-GlcNAcase and α1-6 Mannosidase activities as describedabove. Only α1-6 Mannosidase activity was found in the Q-Sepharosecolumn flow through. Further purification of this enzyme was performedas described below in Section C. Fractions from the Q-Sepharose columnwere assayed for βGlcNAcase activity as described above. The peak ofβ-N-acetylglucosaminidase activity was pooled and eluted from the columnbetween 0.15–0.3 M NaCl. After dialysis against Buffer A overnight, theenzyme pool was applied to a Heparin-TSK (3 ml) column equilibrated withBuffer A. The column was washed with 6 ml of Buffer A followed by alinear gradient of NaCl formed with 45 ml of Buffer A and 45 ml ofBuffer A containing 0.6 M NaCl. Fractions (1.5 ml) were collected at aflow rate of 1 ml/min and assayed for βGlcNAcase activity as describedabove. The peak of enzyme activity was pooled and eluted between0.25–0.3 M NaCl. After dialysis overnight in Buffer A, sodium azide wasadded to 0.02% before storing the enzyme at 4° C. A yield of 30,000units of substantially pure enzyme was obtained after purification ofthe crude extract.

C. α1-6 Mannosidase. The Q-Sepharose flow through described above inSection B was dialyzed overnight in Buffer D (20 mM Potassium phosphatepH 6.0, 10 mM NaCl, 0.1 mM EDTA). After dialysis, the flow through wasapplied to a column of S-Sepharose (1.6×12 cm) equilibrated with BufferD. The column was washed with 40 ml of Buffer D followed by a linearNaCl gradient formed with 80 ml of Buffer D and 80 ml of Buffer Dcontaining 0.6 M NaCl. Fractions (2.5 ml) were collected at a flow rateof 2 ml/min and assayed for α1-6 Mannosidase activity as describedabove. The peak of enzyme activity was pooled and eluted between0.15–0.3 M NaCl. After dialysis overnight against Buffer E (20 mMTris-HCl pH 7.5, 10 mM NaCl, 0.1 mM EDTA), the enzyme pool was appliedto a Heparin-TSK (3 ml) column equilibrated with Buffer E. The columnwas washed with 6 ml Buffer E followed by a linear gradient of NaClformed with 45 ml of Buffer E and 45 ml of Buffer E containing 0.6 MNaCl. Fractions (1.5 ml) were collected at a flow rate of 1 ml/min andassayed for α1-6 Mannosidase activity as described above. The peak ofenzyme activity was pooled and eluted between 0.15–0.2 M NaCl. Afterdialysis overnight in Buffer A, sodium azide was added to 0.02% beforestoring the enzyme at 4° C. A yield of 200,000 units of substantiallypure enzyme was obtained using the above protocol.

D. α1-3,6 Galactosidase. The Heparin pool described above containingα1-3,6 Galactosidase activity was dialyzed overnight against Buffer D.After dialysis, the enzyme pool was applied to column of S-Sepharose(1.6×12 cm) equilibrated with Buffer D. The column was washed with 40 mlof Buffer D followed by a linear gradient of NaCl formed with 80 ml ofBuffer D and 80 ml of Buffer D containing 0.6 M NaCl. Fractions (3 ml)were collected at a flow rate of 1 ml/min and assayed for α1-3,6Galactosidase activity as described above. The peak of enzyme activitywas pooled and eluted between 0.25–0.35 M NaCl. After dialysis overnightin Buffer A, the enzyme pool was applied to a Heparin-TSK (3 ml) columnequilibrated with Buffer A. The column was washed with 6 ml of Buffer Afollowed by a linear gradient of NaCl formed with 45 ml of Buffer A and45 ml of Buffer A containing 1 M NaCl. Fractions (1.5 ml) were collectedat a flow rate of 1 ml/min and assayed for α1-3,6 Galactosidase activityas described above. The enzyme peak was pooled and eluted between0.15–0.25 M NaCl. After dialysis overnight against Buffer A, sodiumazide was added to 0.02% before storing the enzyme at 4° C. A yield of67,500 units of substantially purified enzyme was obtained using theabove protocol.

E. β1-3>>4 Galactosidase. The Phenyl Sepharose pool described above thatcontains both β1-3>>4 Galactosidase and α1-2,3 Mannosidase activitieswas dialyzed overnight against Buffer D. After dialysis, the pool wasloaded onto a column of S-Sepharose (1.0×10 cm) equilibrated with BufferD. The column was washed with 20 ml of Buffer D followed by a lineargradient of NaCl formed with 50 ml of Buffer D and 50 ml of Buffer Dcontaining 0.6 M NaCl. The column flow-through was collected and assayedfor β1-3>>4 Galactosidase and α1-2,3 Mannosidase activity as describedabove. Only α1-2,3 Mannosidase was found in tne S-Sepharose columnflow-through.

Further purification of this enzyme was performed as described inSection F. Fractions (2 ml) were collected at a flow rate of 1 ml/minand assayed for β1-3>>4 Galactosidase activity as described above. Thepeak of enzyme activity was pooled and eluted between 0.15–0.25 M NaCl.After dialysis overnight against Buffer D, the enzyme pool was appliedto a Mono S HR 5/5 (1 ml) column equilibrated with Buffer D. The columnwas washed with 2 ml of Buffer D followed by a linear gradient of NaClformed with 25 ml of Buffer D and 25 ml of Buffer D containing 0.6 MNaCl. Fractions (1 ml) were collected at a flow rate of 1 ml/min andassayed for β1-3>>4 Galactosidase activity as described above. The peakof enzyme activity was pooled and eluted between 0.05–0.1 M NaCl. Afterdialysis overnight against Buffer A, the enzyme pool was loaded onto aHeparin-TSK (3 ml) column equilibrated with Buffer A. The column waswashed with 6 ml of Buffer A followed by a linear gradient of NaClformed with 45 ml of Buffer A and 45 ml of Buffer A containing 0.6 MNaCl. The column flow-through was collected and assayed for β1-3>>4Galactosidase activity. The peak of enzyme activity was found in theflow-through of the column. The flow-through was then dialyzed overnightagainst Buffer A. After the addition of sodium azide to 0.02%, theenzyme was stored at 4° C. The yield of substantially pure enzyme was45,000 units.

F. β1-2,3 Mannosidase. The S-Sepharose column flow-through describedabove that contains α1-2,3 Mannosidase activity was dialyzed overnightagainst Buffer E. After dialysis, the flow-through was applied to acolumn of Q-Sepharose (10×10 cm) equilibrated with Buffer E. The columnflow-through was collected and assayed for α1-2,3 Mannosidase activityas described above. The peak of enzyme activity was found in the columnflow-through. The flow-through was then loaded onto a column of HeparinSepharose CL-6B (1.0×10 cm) equilibrated with Buffer E. The flow thoughwas collected, assayed, and found to contain the peak of enzymeactivity. The flow-through was then loaded onto a Mono Q HR10/10 (8 ml)column equilibrated with Buffer E. The column flow-through and wash wascollected and assayed for α1-2,3 Mannosidase activity described above.The Mono Q column wash was found to contain the peak of enzyme activity.4.36 g of ammonium sulfate was added to the wash with gentle stirring toa final concentration of 1 M. The wash was applied to a column of PhenylSepharose (1.0×10 cm) equilibrated with Buffer B. The column was washedwith 20 ml of Buffer B followed by a linear decreasing gradient ofammonium sulfate formed with Buffer B containing 50 ml of 1 M ammoniumsulfate decreasing to 0.001 M ammonium sulfate. Fractions (2 ml) werecollected at a flow rate of 2 ml/min and assayed for α1-2,3 Mannosidaseactivity as described above. The peak of enzyme activity was pooled andeluted between 0.55–0.3 M (NH₄)₂SO₄. After dialysis overnight againstBuffer D, the enzyme pool was applied to a Poly-Cat A (3 ml) columnequilibrated with Buffer D. The flow-through was collected and assayedfor α1-2,3 Mannosidase activity as described above. The columnflow-through was found to contain the peak of enzyme activity. 4.62 g ofammonium sulfate was added to the flow-through with gentle stirring to afinal concentration of 1 M. The flow-through was then applied to aPhenyl Superose HR10/10 (8 ml) column equilibrated with Buffer B. Thecolumn was washed with 20 ml of Buffer B followed by a decreasing lineargradient of ammonium sulfate formed with 50 ml of Buffer D and 50 ml ofBuffer D containing only 0.001 M (NH₄)₂SO₄. Fractions (1.5 ml) werecollected at a flow rate of 1 ml/min and assayed for α1-2,3 Mannosidaseactivity. The peak of enzyme activity was pooled and eluted between0.65–0.5 M (NH₄)₂SO₄. After dialysis overnight against Buffer E, sodiumazide was added to 0.02% before storing the enzyme at 4° C. A yield of4,000 units was obtained using the above protocol.

G. β-Glucosidase. The Phenyl Sepharose pool described above thatcontains β-Glucosidase activity was dialyzed overnight against Buffer D.After dialysis the pool was applied to a column of S-Sepharose (1.0×10cm) equilibrated with Buffer D. The column was washed with 20 ml ofBuffer D followed by a linear gradient of NaCl formed with 50 ml ofBuffer D and 50 ml of Buffer D containing 0.6 M NaCl. Fractions (1 ml)were collected at a flow rate of 1 ml per minute and assayed forβ-Glucosidase activity as described above. The peak of enzyme activitywas pooled and eluted between 0.1–0.1 M NaCl. After dialysis overnightagainst Buffer D, the enzyme pool was applied to a Mono S HR5/5 (1 ml)column. The column was washed with 2 ml of Buffer D followed by a lineargradient of NaCl formed with 20 ml of Buffer D and 20 ml of Buffer Dcontaining 0.6 M NaCl. Fractions (1 ml) were collected at a flow rate of1 ml/min and assayed for β-Glucosidase activity as described above. Thepeak of enzyme activity was pooled and eluted between 0.05–0.1 M NaCl.After dialysis overnight against Buffer A, the enzyme pool was loadedonto a Heparin-TSK (3 ml) column equilibrated with Buffer A. The columnflow-through and wash was collected and assayed for β-Glucosidaseactivity as described above and the wash was found to contain peak ofenzyme activity. The wash was dialyzed overnight against Buffer A. Afterthe addition of sodium azide to 0.02%, the enzyme was stored at 4° C. Ayield of 500 units was obtained after purification of the crude extract.

H. α1-3,4 Fucosidase. The Phenyl Sepharose wash described above thatcontains α1-3,4 Fucosidase activity was dialyzed against Buffer C. Afterdialysis, the wash was then applied to a column of S-Sepharose (1.0×10cm) equilibrated with Buffer C. The column was washed with 20 ml ofBuffer C followed by a linear gradient of NaCl formed with 50 ml ofBuffer C and 50 ml of Buffer C containing 0.6 M NaCl. Fractions (2 ml)were collected at a flow rate of 2 ml/min and assayed for α1-3,4Fucosidase activity as described above. The peak of enzyme activity waspooled and eluted between 0.15–0.25 M NaCl. After dialysis overnightagainst Buffer C, the enzyme pool was applied to a Mono S HR5/5 (1 ml)column equilibrated with Buffer C. The column was washed with 2 ml ofBuffer C followed by a linear gradient of NaCl formed with 35 ml ofBuffer C and 35 ml of Buffer C containing 0.6 M NaCl. Fractions (1 ml)were collected at a flow rate of 1 ml/min and assayed for α1-3,4Fucosidase activity as described above. The peak of enzyme activity waspooled and eluted between 0.25–0.35 M NaCl. After dialysis overnightagainst Buffer A, the enzyme pool was applied to Heparin-TSK (3 ml)column equilibrated with Buffer A. The column flow-through was collectedand assayed for α1-3,4 Fucosidase activity. The flow-through was foundto contain the peak of enzyme activity. 1.19 g of ammonium sulfate wasadded to the flow-through with gentle stirring to a final concentrationof 1.5 M (NH₄)₂SO₄. The flow-through was then applied to a PhenylSuperose HR10/10 (8 ml) column equilibrated with Buffer F (20 mMTris-HCl pH 7.5, 1.5 M Ammonium sulfate, 0.1 mM EDTA). The column waswashed with 20 ml of Buffer F followed by a decreasing linear gradientof ammonium sulfate formed with 50 ml of Buffer F and 50 ml of Buffer Fcontaining only 0.002 M (NH₄)₂SO₄. Fractions (1.5 ml) were collected ata flow rate of 1 ml/min and assayed for α1-3.4 Fucosidase activity asdescribed above. The peak of enzyme activity was pooled and elutedbetween 0.6–0.5 M (NH₄)₂SO₄. After dialysis overnight against Buffer A,sodium azide was added to 0.02% before storing the enzyme at 4° C. Theyield was 60,000 units obtained after purification of the crude extract.

Purification of α1-3,6 Mannosidase.

280 grams of cells obtained above were suspended in three volumes ofBuffer A′ (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA). The cellsuspension was passed through a Gaulin homogenizer (Model M-15) twice at12,000 psig. The lysate was centrifuged at 13,000 g for 40 minutes in aSharples continuous centrifuge. 700 ml of supernatant were obtained.

The crude extract (700 ml) was loaded onto a column of DEAE SepharoseCL-6B (5.0×26 cm) equilibrated with Buffer A′. The column was washedwith 2500 ml Buffer A followed by a linear gradient of NaCl formed with2000 ml of Buffer A′ and 2000 ml of Buffer A′ containing 1 M NaCl.Fractions (21 ml) were collected at a flow rate of 3 ml/min and assayedfor α1-3,6 Mannosidase activity as described above. The peak of enzymeactivity eluted from the column between 0.15–0.25 M NaCl. Fractionscontaining enzyme activity were pooled and dialyzed against Buffer A′overnight. After dialysis, the enzyme pool was applied to a column ofHeparin Sepharose CL-6B (2.6×25 cm) equilibrated with Buffer A′. Thecolumn was washed with 300 ml of Buffer A′ followed by a linear gradientof NaCl formed with 250 ml Buffer A″ and 250 ml Buffer A′ containing0.95 M NaCl. Fractions (6 ml) were collected at a flow rate of 2 ml/minand assayed for α1-3,6 Mannosidase activity. The peak of enzyme activityeluted between 0.4–0.6 M NaCl. Fractions containing activity were pooledand dialyzed against Buffer A (20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.1mM EDTA) overnight. After dialysis the enzyme pool was applied to acolumn of Q-Sepharose (1.0×10.0 cm) equilibrated with Buffer E (20 mMTris-HCl pH 7.5, 10 mM NaCl, 0.1 mM EDTA). The column was washed with 20ml of Buffer E followed by a linear NaCl gradient formed with 60 ml ofBuffer E and 60 ml of Buffer E containing 0.6 M NaCl. Fractions (2 ml)were collected at a flow rate of 2 ml/min and assayed for α1-3,6Mannosidase activity as described above. The peak of enzyme activity waspooled and diluted between 0.15–0.25 M NaCl. After dialysis in Buffer Eovernight, the enzyme pool was loaded onto a Heparin-TSK (3 ml) columnequilibrated with Buffer E. The column was washed with 6 ml of Buffer Efollowed by a linear NaCl gradient formed with 45 ml of Buffer E and 45ml of Buffer E containing 0.6 M NaCl. Fractions (1.5 ml) were collectedat a flow rate of 1 ml/min and assayed for α1-3,6 Mannosidase activityas described above. The peak of enzyme activity was pooled and elutedbetween 0.1–0.2 M NaCl. After dialysis overnight in Buffer A, sodiumazide was added to 0.02% before storing the enzyme at 4° C. 1000 unitsof substantially pure enzyme were obtained after purification of thecrude extract.

TABLE 5 DETERMINATION OF CO-FACTOR REQUIREMENTS FOR GLYCOSIDASE ACTIVITYENZYME Ca⁺⁺ SUBSTRATE pH β-GlcNAcase GlcNAcβ1-4GlcNAcβ1- 4.5− 4GlcNAc-Coα1-2 Fucosidase Fucαl-2Galβl-4Glc- 6.0− Co α1-3,4 FucosidaseGalβ1-4(Fucα1-3)GlcNAcβ1- 6.0− 3Galβ1-4Glc-Co α1-3,6 GalactosidaseGalα1-3Galβ1-3GlcNAc- 6.0+ Co β1-3>>4 GalactosidaseGalβ1-3GlcNAc1-3Galβ1- 4.5− 4Glc-Co β-Glucosidase Glcβ1-4Glcβ1-4Glc-4.5+ Co α1-2,3 Mannosidase Manα1-3Manβ1-4GlcNAc- 6.0+ Co α1-6Mannosidase Galβ1-4GlcNAcβ1-2Manα1- 4.5− 6Manβ1-4GlcNAc-Co α1-3,6Mannosidase Manα1-3Manβ1-4GlcNAc- 6.0+ Co β-Xylosidase Manα1-6(Manα1-3)(Xylβ1- 6.0+ 2) Manβ1-4GlcNAcβ1-4(Fucα1- 3)GlcNAc-Co β-MannosidaseManβ1-4Manβ1-4Man- 5.5− Co

Example 4 Characterizing Glycosidases

A. Fucosidases.

Reaction conditions were optimized for α1-3,4 Fucosidase (I) and α1-2Fucosidase (II). 1.0 nmol of substrate in 50 mM sodium citrate pH 6.0was digested with enzyme (2 units of α-Fucosidase II (FIG. 4, lane 2),100 units of (α-Fucosidase I (FIG. 4, lane 3); 2 units of (α-FucosidaseI (FIG. 4, lanes 5, 6, 9, 10); 20 units (α-Fucosidase II (FIG. 4 lanes7, 11); and 2 units of β-Galactosidase (FIG. 4, lanes 6, 10) (Bovinetestes, BMB) (1 unit is defined as the amount of enzyme required torelease 1 nanomole of terminal sugar from an oligosaccharide at 37° C.in 1 hour). No cofactors were found to be necessary. The incubation wasperformed at 37° C. for 4 hours and for 24 hours. At 4 hours, thedigestion of substrate with Xanthomonas manihotis Fucosidases wascomplete. The incubation was extended to 24 hours so as to permit theless active β-Galactosidase digestion to be accomplished.

FIG. 4 shows substrate specificity of (α-Fucosidases type I and IIisolated from Xanthomonas manihotis that differ in their specificity forselected glycosidic linkages. α-Fucosidase I selectively cleavesFucα1-3R and Fucα1-4R linkages, and α-Fucosidase II selectively cleavesFucα1-2R linkages as demonstrated by cleavage of coumarin labelledoligosaccharide substrates and separation of the reaction products bythin layer chromatography (lanes 3, 5, 9). The α-Fucosidase II cleaved aterminal α1-2 linkage on a trisaccharide as shown by the migration ofthe band on the TLC to a higher position corresponding to the loss ofone monosaccharide (lane 2) but did not recognize branched Fucα1-3R orFucα1-4R linkages (lanes 7, 11). In contrast to α-Fucosidase II,α-Fucosidase I was unable to digest Fucα1-2R linkages.

To confirm that the α1-3,4 Fucosidase I removed the fucose and not theterminal galactose, the substrates were digested with α-Fucosidase I and0.5 units of bovine testes β-Galactosidase (BMB) to remove both terminalβ-galactose and fucose from the substrates. In FIG. 4, lanes 6 and 10show that a second monosaccharide (terminal galactose) was removed aftertreatment with α-Fucosidase I and β-Galactosidase, whereas in lanes 5and 9 only one monosaccharide was removed following α-Fucosidase Idigestion.

Substrate specificity was demonstrated using three substrates: 120:Fucα1-2Galβ1-4Glc-Co; 95: Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-Co; and113: Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc-Co. The controls woreundigested substrate (lanes 1, 4, 8).

TABLE 6 α-FUCOSIDASES I AND II SUBSTRATE III 120: Fucαl-2Galβ1-4Glc-Co−+ 95:

+− 113:

+− I: α1-3,4 Fucosidase II: α1-2 FucosidaseB. β-N-Acetylglucosaminidase.

10 units of β-N-Acetylglucosaminidase (β-GlcNAcase) purified as abovefrom Xanthomonas manihotis was found to react with 0.5–1 nmol substratein the absence of cofactors in 50 mM sodium citrate pH 4.5 although theenzyme was similarly active at a pH in the range of pH 4–6. Incubationwas carried out for 4 hours at 37° C.

FIG. 5 shows the results of these reactions analyzed by TLC. Lanes 1 and3 are undigested substrates of 118 (GlcNAcβ1-4GlcNAcβ1-4GlcNAc-Co) and167 (Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Co) respectively. Lane 2 shows theeffect of cleaving at the terminal GlcNAcβ1-4R linkage. Lane 4 showscleavage of the terminal Galβ1-3R linkage using 0.5 units of bovinetestes β-Galactosidase and lane 5 shows the additional cleavage ofGlcNAcβ1-3R linkage with β-N-Acetylglucosaminidase after cleavage withβ-Galactosidase.

FIG. 6 further characterizes the specificity of the β-Nacetylglucosaminidase for β-GlcNAc using substrates containingGlcNAcβ1-2R, GlcNAcβ1-3R and GlcNAcβ1-6R linkages, the latter forming abranchpoint (200: Galβ1-4GlcNAcβ1-2Manα1-6Manβ1-4GlcNAc-Co; 197:Galβ1-4GlcNAcβ1-6 (Galβ1-4 GlcNAcβ1-3) Galβ1-4Glc-Co). These substrateshave a terminal Galβ1-4 residue which is cleaved with β-Galactosidase(bovine testes) prior to cleavage with Xanthomonas manihotisβ-GlcNAcase. Lanes 1 and 4 show uncleaved substrates 200 and 197respectively while lane (m) has a disaccharide and tetrasaccharide assize markers. Lanes 2 and 5 are substrate 200 and 197 digested withβ-Galactosidase respectively and lanes 3 and 6 are substrates 200 and197 respectively digested with β-Galactosidase and β-GlcNAcase.

FIG. 7 shows that β-GlcNAcase from Xanthomonas manihotis does not digestβ-N-acetylgalactosamine (β-GalNAc) whereas β-GlcNAcase from bovinekidney does. Lanes 1 and 4 contain undigested substrate 96(GalNAcβ1-3Galα1-4Galβ1-4Glc-Co) and 205 (GalNAcβ1-4Galβ1-4Glc-Co)respectively. Lanes 2 and 5 are substrates 96 and 205 respectivelydigested with β-GlcNAcase from Xanthomonas manihotis. No cleavagereaction is detected. However, with β-GlcNAcase from bovine kidney(lanes 3 and 6), cleavage of β-GalNAc is seen for both substrate 96 and205. A marker is included (m) consisting of a disaccharide and atetrasaccharide that shows cleavage by bovine kidney β-GlcNAcase of asingle monosaccharide for each of 96 and 205.

The results are summarized in Table 7. Only β-GlcNAc from Xanthomonashad no detectable activity for PNP-GalNAc whereas the commerciallyavailable enzymes did have activity for this substrate.

TABLE 7 COMPARISON OF HEXOSAMINIDASE ACTIVITIES ON PNPβGlcNAc v.PNPβGalNAc PNPβGlcNAc PNPβGalNAc SOURCE OD 400 OD 400 Xanthomonasmanihotis >2.0 ND* Streptococcus pneumoniae >2.0 >2.0 Chickenliver >2.0 >2.0 Bovine kidney >2.0 >2.0 Assays were performed using 1NEB unit of enzyme at 37° C. for 1 hour as defined in Example 2. ND =not detectable PNP substrate was 10 mM, reaction volume was 25 μl,reaction was stopped using 75 μl 0.2M sodium borate pH 9.8, and theresulting absorbance was read at OD 400. *50 NEB units of the enzymepurified from Xanthomonas manihotis was assayed on PNPβGalNAc. (Evenwhen incubated overnight, no measurable activity on PNP-GalNAc could bedetected.)C. Mannosidase.Specificity for Linear Structures.

Three enzymes isolated from Xanthomonas manihotis were characterized fortheir substrate specificity using coumarin-labelled oligosaccharides.The substrates are listed in Table 8 and cleavage is recorded by a (+).The TLC data from which this Table is derived is shown in FIG. 10. Themarker shown on the TLC is a mixture of oligosaccharides consisting ofdisaccharides, tetrasaccharides and hexasaccharides. Lanes 1, 7 and 11are undigested substrates.

Digestions were performed utilizing 1 nmole substrate prepared in abuffer of 50 mM sodium citrate pH 6.0 with enzyme concentrationsdescribed below. 5 mM Ca++ was added to the incubation mixture forα1-3,6 Mannosidase (I) and α1-2,3 Mannosidase (III).

Lanes 2, 8, and 14 show the digestion products if any of Mannosidase Ifor substrates 134, 114, and 200 respectively. This enzyme is unable tocleave the terminal Manα1-2R linkage on substrate 134 even in thepresence of a relatively high concentration (15 units) of enzyme and aprolonged incubation period (20 hours). In contrast, the enzyme iscapable of cleaving the terminal Manα1-2R linkage on substrate 114 andthe terminal Manα1-6R linkage on substrate 200, the latter afterGalβ1-4GlcNAcβ1-2 has been removed. Cleavage in these examples occursusing only 1.5 units of enzyme incubated for 2 hours with substrate.

Lanes 5, 10, and 16 show the digestion products of α-Mannosidase II.This enzyme does not cleave Manα1-2R or Manα1-3R linkages even atconcentrations of 100 units incubations for 20 hours. In contrast, theenzyme does cleave Manα1-6R linkage from substrate 200 after removal ofGalβ1-4GlcNAcβ1-2.

Lanes 3, 4, 9 and 15 show the digestion products if any of α-MannosidaseIII. This enzyme cleaves the terminal Manα1-2R linkage on substrate 134,and the terminal Manα1-3R linkage on substrate 114 but does not cleavesubstrate 200 even after Galβ1-4GlcNAc β1-2 has been removed. Wherecleavage is observed, 1.5 units of enzyme is incubated for 2 hrs withsubstrate. Where no activity is observed, 15 units of enzyme were usedfor 20 hours.

In keeping with the above cleavage activities, the α-Mannosidase I hasbeen identified as α1-3,6 Mannosidase; α-Mannosidase II has beenidentified as α1-6 Mannosidase and α-Mannosidase III has been identifiedas α1-2,3 Mannosidase

TABLE 8 α-MANNOSIDASES I, II, III SUBSTRATE I IIIII 134:Manα1-2Manα1-3Manβ1-4GlcNAc-Co − −+ 114: Manα1-3Manβ1-4GlcNAc-Co + −+200: Manα1-6Manβ1-4GlcNAc-Co + +− I: α1-3,6 Mannosidase II: α1-6Mannosidase III: α1-2,3 MannosidaseSpecificity for Branched Structures.

Incubation of α-Mannosidase I (2 hrs.) against substrates 213 and 216 asshown in FIG. 11 revealed that this enzyme is capable of cleavingbranched structures. Lanes 2 and 8 show removal of 2 mannose residues.Further incubation (20 hours) resulted in the removal of the second pairof branched mannoses (lanes 13, 19). Whereas this digestion was partial,possibly because of the negative effect of the neighboring labelledmannose at the reducing end, it is likely that the second branch wouldhave been cleaved in a naturally occurring oligosaccharide substrate.

Incubation of α-Mannosidase II (2 hrs and 20 hours) showed no evidenceof cleavage of substrate 213 and 216. (lanes 6, 11, 17 and 23). Whilenot wishing to be bound by theory, it is conjectured that α-MannosidaseII (α1-6 Mannosidase) is capable of cleaving linear molecules but not ofcleaving branched molecules. Use of this enzyme can provide a means ofdifferentiating branched and linear Manα1-6R glycosidic linkages.

Incubation of α-Mannosidase III (2 hours and 20 hours) with substrates213 and 216 (lanes 4, 9, 15, and 21) resulted in partial cleavage of thesubstrates with the removal of a single mannose from 216 consistent withthe enzyme specificity for Manα1-2R and Manα1-3R. The results indicatethat this enzyme has a greater affinity for linear substrates than forbranched substrates. In the presence of α-Mannosidase II, someadditionalcleavage was observed for substrate 213 that was not apparentin substrate 216.

D. α1-3,6 Galactosidase.

As shown in FIG. 9, α1-3,6 Galactosidase preferentially cleaves Galα1-3Rand Galα1-6R linkages (lanes 3, 6 and 9) where lane 3 shows release ofone monosaccharide with substrate 109 (Galα1-3Galβ1-3GlcNAc-Co), and amonosaccharide cleavage product with substrate 181(Galα1-6Glcα1-2Fru-Co) when 10 units of enzyme are added to 1 nmole ofsubstrate in 50 mM Na citrate pH 6.0 supplemented with 5 mM CaCl₂ andincubated for 2 hours at 37° C. In contrast, the enzyme does not cleavesubstrate 193 (Galα1-4Galβ1-4Gal-Co) as shown in lane 5 even when theconcentration of the enzyme is increased to 100 units compared with 10units and the incubation time is increased to 20 hours compared to 2hours. These results contrast with a commercially availableα-Galactosidase derived from coffee bean (BMB). The coffee beanα-Galactosidase readily cleaves substrate 193 at the Galα1-4R linkage asshown in lane 6. Undigested substrate without enzyme is included as acontrol in lanes 1, 4, and 7. Markers are included that contain adisaccharide, a tetrasaccharide, and a hexasaccharide.

E. β1-3>>4 Galactosidase.

As shown in FIG. 8, β1-3>>4 Galactosidase preferentially cleavesGalβ1-3R linkages where lane 2 shows release of at least onemonosaccharide with substrate 167 (Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Co), when1 unit of enzyme is added to 1 nmole substrate in 50 mM Na citrate pH4.5 and incubated for 2 hours at 37° C. In contrast, the enzyme onlypartially cleaves substrate 202 (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-Co) asshown in lane 6 when the concentration of the enzyme is increased to 100units compared with 1 unit. These results contrast with a commerciallyavailable β-Galactosidases derived from chicken liver (cl) and bovinetestes (bt) both of which cut substrates 167 and 202 equally well (lanes3, 4, 7, 8). Undigested substrate without enzyme is included as acontrol in lanes 1 and 5.

F. β-Glucosidase.

As shown in FIG. 12, a crude extract derived from Xanthomonas manihotiswas found to have a specificity for at least Glcβ1-4R linkages. Enzymedigestions were performed at 37° C. for 2 hrs. in an incubation mixturecontaining 1 nmole substrate in 50 mM Na citrate pH 4.5 and 5 mM CaCl₂.Various concentrations of enzyme were used. The reaction mixtures werethen spotted onto TLC plates. Substrate without enzyme was added asnegative controls and additional positive controls were added whichconsisted of substrate and βGlcNAcase (lane 7) and substrate andβ-Galactosidase (Bovine testes) (lane 10). Lanes 2, 4, 6, and 9 show thereaction products when 1 unit of β-Glucosidase (β-Glcase) is mixed with179 (Glcβ1-4Glcβ1-4Glc-Co); 5 units of β-Glcase is mixed with 180(Glcα1-4Glcα1-4Glc-Co), 5 units of β-Glcase is mixed with 118(GlcNAcβ1-4GlcNAcβ1-4GlcNAc-Co) and 5 units of β-Glcase is mixed with202 (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-Co). Of the four substrates tested,only substrate 179 containing a terminal Glcβ1-4 linkage is cleaved bythe β-Glcase.

Example 5 Cloning of Exoglycosidase Genes

The method for cloning of exoglycosidase genes is described usingXanthomonas as the source of naturally occurring glycosidase. However,the method may be applied not only to this embodiment but to anyorganism in which the probability of finding at least one glycosidasehas been determined as described above.

A. DNA Purification.

To prepare the DNA of Xanthomonas manihotis, 1 g of cell paste wasresuspended by shaking gently for 30 min in 5 ml of 0.1 M Tris-HCl, 0.1M EDTA pH 7.6. The suspension was divided into two 3.0 ml portions. 3.5ml of 1.7 mg/ml lysozyme in 0.1 M Tris-HCl, 0.1 M EDTA pH 7.6 was addedto each portion and each was incubated for 15 minutes at 37° C. SDS wasadded to 1%, and proteinase K was added to 0.13 mg/ml and then theportions were incubated for 1 hour at 37° C. 0.4 ml of a solution of 10%SDS and 8% sarcosyl was added to each and incubation was continued at55° C. for 2 hours. The two portions were then combined and dialyzedagainst four changes of DNA buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0)for 24 hours. The dialyzed DNA solution was then prepared for cesiumchloride-ethidium bromide equilibrium density centrifugation byincreasing the total volume to 40 ml with DNA buffer, and then dividingthe DNA solution into two 20 ml portions, to each of which 20 grams ofcesium chloride and 0.2 ml of 5 mg/ml ethidium bromide were added. TheDNA solution was centrifuged at 44,000 rpm for 48 hours and theresulting band of DNA was removed with a syringe and an 18 gauge needle.The ethidium bromide was removed by extracting 4 times with an equalvolume of ice-cold, water-saturated N-butanol. The cesium chloride wasremoved by dialysis. The DNA was then precipitated by adding NaCl to0.5M and layering 0.55 volume isopropyl alcohol on top. The precipitatedDNA was spooled onto a glass rod. The DNA was dissolved in 2 ml 10 mMTris, 1 mM EDTA pH 8.0 to a final concentration of approximately 76μg/ml.

B. Partial Digestion.

The purified DNA was cleaved with Sau3AI to achieve partial digestion asfollows: 124 μl of DNA at 76 μg/ml in 100 mM Bis Tris Propane-HCl pH7.0, 10 mM MgCl₂, 100 mM NaCl, 1 mM dithiothreitol buffer with 100 μg/mlBSA was divided into one 400 μl aliquot and four 200 μl aliquots. To the400 μl tube was added 2 units of Sau3AI to achieve 1 unit of enzyme/4.75μg of DNA. 200 μl was withdrawn from the first tube and transferred tothe second tube to achieve 0.5 units Sau 3AI/4.75 mg and so on, eachsucceeding tube receiving half of the previous amount of Sau3AI. Thetubes were incubated at 37° C. for 15 minutes, heat-treated at 72° C.for 15 minutes then subjected to electrophoresis in a 0.7% agarose gelin Tris-Borate-EDTA buffer. DNA fragments ranging in size from about 9to 2 kb were collected by electrophoresing into DEAE anion exchange ofpaper for 2 hr. The paper was washed two times with 150 μl of buffercontaining 0.1M NaCl, 10 mM Tris pH 8.0 and 1 mM EDTA. Subsequently, theDNA was eluted from the paper by washing the paper four times with 75 μlof a buffer containing 1.0 M NaCl, 10 mM Tris pH 8.0 and 1 mM EDTA. Theresulting solution containing the DNA fragment was extracted with 300 μlphenol/chloroform followed by extraction with 300 μl chloroform andprecipitated with 1 ml absolute ethanol by placing in a dry ice/ethanolbath for 15 min. The DNA was pelleted at 14K rpm for 5 min. The pelletwas rinsed with 70% ethanol, air dried and resuspended in a final volumeof 10 μl 10 mM Tris pH 8, and 1 mM EDTA. The purified fragments wereused as described in step 3 below.

C. Ligation.

The fragmented DNA was ligated to pUC19 as follows: 3 μg ofSau3AI-partially digested Xanthomonas manihotis DNA (10 μl) was mixedwith 1.5 μg of BamHI-cleaved and dephosphorylated pUC19 (1 μl). 4 μl of10× ligation buffer (500 mM Tris pH 7.5, 100 mM MgCl₂, 100 mM DTT, 5 mMATP) was added, plus 25 μl of sterile distilled water to bring the finalvolume to 39 μl. One μl of concentrated T4 DNA ligase (2×10⁶ U/ml) wasadded and the mixture was incubated at 37° C. for 2 hours. Ten μl of theligation mixture was deionized by drop dialysis using a Millipore VS0.025 μM filter. The DNA was then electroporated into E. coli ED8767.The E. coli was prepared for electroporation by growing up 1 L of cellsto Klett 50–80 in L-broth. The cells were chilled on ice for 15 to 30minutes and then pelleted in the cold at 4,000 rpm for 15 mins. Thepellet was washed 2 times in ice cold sterile water and once in 10%glycerol. The washed pellet was resuspended in 1 to 2 ml of 10% glycerolto a final cell concentration of 3×10¹⁰ cells per ml. The cells werefrozen until needed in 100 μl aliquots at −70° C. To electroporate theDNA into the prepared cells, the cells were gently thawed and placed onice. 40 μl of cells were mixed with 10 μl of the ligated and dialyzedDNA. The mixture was placed into a cold 0.2 cm electroporation cuvette.A pulse of electricity at 12.5 kv/cm with a time constant of 4–5 msecwas applied to the DNA cell mixture. The E. coli was immediately dilutedwith 1 ml L-broth, allowed to grow at 37° C. for 30 min. and plated on150 mm L-agar plates containing selective media. After overnightincubation at 37° C., clones expressing an exoglycosidase were screenedas described below.

D. Screening for Exoglycosidase Clones.

To screen for clones which express exoglycosidase activities threedifferent chromogenic indicator substrates were employed. Onechromogenic substrate used was5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside (X-gal) which was addedto the selective agar plates at a concentration of 50 μg/ml before thetransformed cells are plated on the agar. Any colony grown on agarplates containing X-gal which expresses a β-Galactosidase will be blue.Of the 9×10⁴ colonies screened in this manner only one colony was blue.The other type of chromogenic substrates used to screen forexoglycosidase activity was the 4-methylumbelliferyl (4-MU) substrates.These substrates were either sprayed on the surface of the selectiveplate at a concentration of 1 μg/ml after colonies have formed or addedto 1.5% agar and used to overlay the selective plate containingcolonies. After spraying or overlaying colonies producing activeexoglycosidase were identified by viewing the colonies with long waveultraviolet light (366 nm). In this experiment a mixture of 4-MUsubstrates was added to a 1.5% agar overlay. Those included in the mixwere: 4-MU-N-acetyl-β-D-glucosaminide, 4-MU-β-D-mannopyranoside,4-MU-α-D-glucoside, 4-MU-β-D-glucoside, and 4-MU-α-D-galactoside. Anycolony producing an active β-N-Acetylglucosaminidase, β-Mannosidase,α-Glucosidase, β-Glucosidase, or α-Galactosidase which is able to cleaveone of these substrates will fluoresce blue under UV light. Of the 2×10⁴colonies screened using these substrates, 8 fluorescent colonies wereisolated. Three isolates were determined to be clones producingα-Galactosidase, two produced β-Glucosidase, one producedβ-N-Acetylglucosaminidase, and the remaining two had no detectableexoglycosidase activity as determined by their ability to cleave theirrespective 4-MU substrates as well as their respective p-nitrophenylsubstrates.

Other methods have to be employed to screen for clones that expressexoglycosidase activities but do not cleave synthetic substrates. Toscreen for clones expressing the α-Mannosidases I or II from Xanthomonasmanihotis a differential plating medium was used. The medium used is avariation on EMB agar which was used to screen for E. coli mutants whichwere unable to utilize lactose as a carbon source (lac⁻). TraditionalEMB agar contains lactose and two indicator dyes, eosin yellow andmethylene blue. When a strain of E. coli which is capable of fermentinglactose (lac⁺) grows on EMB agar the colonies appear dark purple toblack; however, lac colonies are white due to their inability to fermentlactose. To screen for clones expressing an α-Mannosidase the library(described above) was plated at a concentration of approximately 30,000cfu/ml on M9 minimal media containing 100 mg carbenicillin, 0.4 g eosinyellow, 0.065 g methylene blue and 1 g α-mannobiose per liter of medium.The disaccharide mannobiose (mannose α1-6 mannose) cannot be utilized asa carbon source by E. coli unless they express the cloned α-Mannosidasewhich will cleave the mannobiose into mannose which can then befermented by the host. The plates were incubated at 37° C. for 2–4 days.Eleven colonies out of 15,000 cfu plated were pigmented deep purple.These colonies were picked, streaked for isolated colonies on LB agarcontaining 100 μg/ml ampicillin and grown at 37° C. Of the 11 coloniestested, crude extracts from two showed α1-3,6 Mannosidase (Man I)activity. No clones were isolated which expressed the other Mannosidasesfrom Xanthomonas manihotis (α1-2, 3 and α1-6 specific) This method canbe used to isolate other exoglycosidases which will not cleave syntheticsubstrates. The only limitations are: 1) that the di-, tri-, oroligosaccharide cannot be utilized by the host unless the exoglycosidaseis present, 2) that the sugar released by the exoglycosidase must beable to be utilized by E. coli as the sole carbon source, and 3) thatthe sugar substrate must be available in sufficient quantities that itcan be added to the agar base to allow growth of the host expressing theexoglycosidase.

Although certain preferred embodiments of the present invention havebeen described, the spirit and scope of the invention is by no meansrestricted to what is described above. For example, within the generalmethod for isolating glycosidases from Xanthomonas, it is also possibleto isolate endoglycosidases wherein the cell extracts are screenedagainst appropriately labelled substrates. In addition, within thegeneral method for cloning glycosidases, endoglycosidases may be clonedusing appropriately labelled substrates.

Example 6 Preparation of Substrates For Enzymes Assays: AMC-Labeling ofOligosaccharides

Substrates were either purchased from Accurate Chemical and ScientificCorp. (Westbury, N.Y.), Pfanstiehl Labs (Waukega, Ill.), V-Labs, Inc.,(Covington, La.) or isolated according to the method incorporated byreference from Carbohydrate Analysis: A Practical Approach (1986) Eds.Chaplin, M. F. Kennedy J. F. (IRL Press Limited, England) pp. 150–151).Silica Gel 60 preparative plates (1000 μm thick 20×20 cm) were obtainedfrom EM Science (Gibbstown, N.J.). 7-aminomethylcoumarin (AMC) wasobtained from Eastman Kodak (Rochester, N.Y.). Horseradish peroxidasewas purchased from Sigma Chemical Company (St. Louis, Mo.).

Oligosaccharides (0.1–5.0 μmoles) were dissolved in 100 μl H₂O. Theaqueous carbohydrate solution was added to a solution containing 300 μlmethanol, 20 mg 7-amino methylcoumarin (AMC), 35 mg NaCNBH₃ and 41 μlglacial acetic acid. The mixture was sealed into a screw cap microfugetube and heated in a dry block at 85° C. for 45 minutes. The reactionwas loaded onto a Sephadex G-25 column (2×50 cm) equilibrated with H₂O.The product was eluted with H₂O and 1 ml fractions were collected. Afterassaying fractions for purity by TLC as described below, appropriatefractions were pooled and vacuum concentrated to 0.1–1 μmol/ml beforestorage at −20° C.

Oligosaccharide obtained from horseradish peroxidase was released fromthe protein by hydrazinolysis and reacteylated (A Carbohydrate Analysis:A Practical Approach (1986) Eds. Chaplin, M. F. Kennedy J. F. (IRL PressLimited, England) supra). After desalting carbohydrate on a SephadexG-25 column (2.5×30 cm), fractions were assayed by a modified neutralsugar (Dubois, et al., 1956, Anal. Chem., 28:350–356) using a microtiterplate format. Briefly, samples were adjusted to a final volume of 90 μlwith H₂O. 5 μl of 85% phenol/H₂O (v:v) was added followed by the rapidaddition of 180 μl H₂SO₄. The sugar concentration was determined byreading the absorbance at OD₄₉₀. Various concentrations of mannose wasused to generate a standard curve. Oligosaccharide-containing fractionswere pooled and labeled as described above. Following Sephadex G-25chromatography, the sample was further purified using AbsorptionPreparative Layer Chromatography by streaking the sample onto a 1000 μmthick 20×20 cm Silica Gel 60 preparative plate. Following chromatographyin isopropanol:ethanol:H₂O (2.3:1.0:0.7, v:v:v), the appropriate bandwas excised and the silica crushed. The carbohydrate was eluted bywashing the silica with 50% isopropanol:water (v:v) until the eluant nolonger emitted fluorescence. The eluant was vacuum concentrated to 0.1μmol/ml before storage at −20° C. The oligosaccharide structure(Kurosaka, J. Biol. Chem. (1991) 266:4168–4172) was confirmed byexoglycosidase digestion and TLC analysis as described below.

Example 7 Method For Screening Organisms For Glycosidase Activity

Silica gel 60 without F glass backed TLC plates were purchased from EMScience (Gibbstown, N.H.). p-nitrophenyl glycopyranosides were purchasedfrom Sigma Chemical Co. (St. Louis, Mo.). Control glycosidases wereobtained from Boehringer Mannheim (Indianapolis, Ind.), OxfordGlycoSystems (Rosedale, N.Y.) or Seikagaku (Rockville, Md.). Columns andchromatography reagents were purchased from Pharmaia (Piscataway, N.Y.)or TosoHaas (Montgomeryville, Pa.).

Preparation of Cell Extracts For Screening Assay.

Cell pastes (0.1–0.5 g) were thawed and suspended in three volumes ofBuffer A′ (20 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1 mM Na₂EDTA). Afterbeing briefly sonicated, the cell suspensions were centrifuged at 14,000rpm for 10 minutes at 4° C. in an Eppendorf microcentrifuge.

Glycosidases Digestion Reaction.

Cell extracts, cell growth media or partially-purified extracts wereassayed for glycosidase activity by adding 1–5 μl to a 10 μl mixturecontaining 1 nmole of AMC-labeled oligosaccharide in 50 mM sodiumcitrate pH5.5 (various pH's and cofactors, see Table 5). Afterincubations at 37° C. for a period in the range of 5 minutes to 20hours, the reactions were analyzed by TLC as described below. Toquantitate the final yield of β-xylosidase activityXylβ1-4Xylβ1-4Xylβ1-4Xyl-Co was used as a substrate in 50 mM sodiumcitrate pH4.5. One unit of β-xylosidase was defined as the amount ofenzyme required to release 1 nmol of terminal xylose from theoligosaccharide substrate at 37° C. in 1 hour. To quantitate the finalyield of β-mannosidase activity, Manβ1-4Manβ1-4Man-Co was used as asubstrate in 50 mM sodium citrate pH5.5, supplemented with 100 μg/mlbovine serum albium. One unit of β-mannosidase was defined as the amountof enyzme required to release 1 nmol of terminal mannose from theoligosaccharide substrate at 30° C. in 1 hour.

Analysis of Digestion Products By Thin Layer Chromatography (TLC) ofAMC-labeled Oligosaccharides.

A small volume of glycosidase digestion reaction (2–3 μl) was spotted ina tight band on a silica gel TLC glass-backed plate. The bands werecompletely dried with a hot air gun (temperature not exceeding 70° C.The plate was developed until the solvent front moved 10 cm inisopropanol:ethanol:H₂O (2.5:1.0:0.5, v:v:v). Following chromatography,the AMC-substrates, which remained near the origin, and their hydrolyzedproducts, which migrated upward with the mobile phase, were visualizedwith a 314 nm UV lamp. Controls included markers of undigesteddisaccharide (Galβ1-4GlcNAc-Co), tetrasaccharide(Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Co) and hexasaccharide(Galβ1-4GlcNAcβ1-6[Galβ1-4GlcNAcβ1-3]Galβ1-4G Undigested substrates alsoserved as controls.

The results of screening cell extracts againstManα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4GlcNAcβ1-4 (Fucα1-3)GlcNAc-Cofrom different bacterial strains of Xanthomonas and Bacillus are shownin FIG. 13 (see, Tables 3 and 4). Although cell extracts from Bacillusstrains are demonstrated no activity, the cell extract from one strain,X. manihotis, was capable of removing three sugars from theAMC-substrate. Cell extracts from two other strains X. holicola and X.oryzae were capable or removing four sugars suggesting the presence ofα-Mannosidase, β-Xylosidase and either α-Fucosidase or β-Mannosidaseactivities.

Example 8 Screening Organisms Using p-nitrophenyl Glycoside Substrates

Exoglycosidases have typically been identified and purified usingderivatized monosaccharides such as p-nitrophenyl glycopyranoside assubstrates (Hayward, A.C. (1977) J. Appl. Bacteriol. 43:407–411) Thesesubstrates provide information about an enzyme's ability to recognizespecific monosaccharides including their anomericity. However, noinformation is obtained regarding an enzyme's linkage specificitybecause the monosaccharide is not linked to a second sugar, but ratherchemically linked to a chromogenic marker. Often glycosidases capable ofcleaving a monosaccharide derivative fail to hydrolize the sugar residuewhen it is part of an oligosaccharide (Talbot, G. and Sygusch, J. (1990)Appl. Environ. Microbiol. 56:3505–3510).

As demonstrated by Hayward (supra), Xanthomonas campestris strains willcleave several p-nitrophenyl glycopyranoside substrates. Cell extracts(5 μl) prepared as in Example 2 were incubated with 25 μl of 50 mMsodium citrate pH 5.5 containing 250 nmol of either p-nitrophenylβ-D-galactopyranoside, p-nitrophenyl α-D-glucopyranoside, p-nitrophenylβ-D-glycopyranoside, p-nitrophenyl β-D-xylopyranoside or p-nitrophenylN-acetyl-β-D-glucosaminide for 4 hours at 37° C. The reaction wasstopped by adding 75 μl of sodium borate pH9.8 and the absorbance of thereaction mixture was measured at 410 nm. The results using thisscreening assay on two X. campestris strains, NEB420 and NEB497, isshown in FIG. 14. Both strains cleaved all p-nitrophenyl glycopyranosidesubstrates tested suggesting the presence of β-galactosidase,α-glucosidase, β-glucosidase, β-xylosidase and β-N-acetylglucosaminidaseactivities.

However, when using AMC-substrates as described in Example 2, the cellextracts from the two strains demonstrated only α- and β-glucosidaseactivities as shown in FIGS. 14 and 15. Cell extracts (5 μl were accedto a 10 μl reaction mixture containing 1 nmol of AMC-substrate in 50 mMsodium citrate pH 5.5 at 37° C. for 4 hours and analyzed by TLC asdescribed in Example 2. FIG. 15 shows the results of cell extractsNEB420 and NEB497 tested on 202: Galβ1-4GlcNAcβ1-3Galβ1-4Glc-Co (lanes 2and 3, respectively), 167: Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Co (lanes 5 and6, respectively), 180: Glcα1-4Glcaα1-4Glc-Co (lanes 8 and 9,respectively), 179: Glcβ1-4Glcβ1-4Glc-Co (lanes 11 and 12, respectively)and 233: GlcNAcβ1-4GlcNAcβ1-4GlcNAc-Co (lanes 14 and 15, respectively).Undigested substrates (lanes 1, 4, 7 and 10) served as controls. In FIG.15; only lanes 8, 93 11 and 32 indicate the release of a terminalmonosaccharide. Lanes 8 and 9 show the removal of α-glucose while lanes11 and 12 show the release of β-glucose demonstrating the presence of α-and β-glucosidase activities in both strains. All other AMC-substrateswere resistant to hydrolysis when incubated with cell extracts from thetwo X. campestris strains thereby demonstrating that glycosidaseactivity as measured on specific monosaccharide derivatives do notalways translate to activity on that sugar residue when it is part of anoligosaccharide. Similarly, glycosidases discovered for their ability tocleave oligosaccharide substrates do not always hydrolyze a derivatizedmonosaccharide (Sano, et al., (1992) J. Biol. Chem. 267:1522–1527).

Example 9 Method For Purification of Glycosidases From XanthomonasHolcicola

Fermentation of Xanthomonas holcicola.

Xanthomonas hocicola strain NEB121 (ATCC #13461) was grown in mediaconsisting of 10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl and 0.3g/l NaOH. The cells were incubated at 30° C. until late logarithmicstage with aeration and agitation. The cells were harvested bycentrifugation and stored frozen at −70° C.

Preparation of Crude Extract.

All further procedures were performed either on ice or at 4° C. Cellpast (191 g) obtained above was suspended in two volumes of Buffer A (20mM Tris-HCl (pH7.51, 50 mM NaCl, 0.1 mM Na₂EDTA). The cell suspensionwas passed through a Gaulin homogenizer (Model M-15) twice at 12,000psi. The lysate was centrifuged at 1,300 g for 40 min. in a Sharplescontinuous centrifuge. 565 ml of supernatant was obtained.

Purification of Glycosidases.

Glycosidases were separated and purified from crude cell extracts byusing a series of separation methods that differentiated the enzymesaccording to their hydrophobicity and their charge. Enzymes were assayedaccording to the methods described in Example 7 using conditionsdescribed in Table 5.

The crude extract (565 ml) was loaded onto a column of DEAE SepharoseCL-6B (5.0×15 cm) equilibrated with Buffer A. The column flowthrough,containing both the β-Xylosidase and β-Mannosidase, was applied to acolumn of Heparin Sepharose CL-6B (2.6×15 cm) equilibrated with BufferA. The column was washed with 160 ml of Buffer A followed by a lineargradient of NaCl (0.05–0.95 M) in 400 ml of Buffer A (flow rate, 1ml/min; 8 ml fractions). Fractions containing β-Xylosidase that elutedwith the NaCl gradient (0.35–0.6M) were pooled and the enzyme furtherpurified as described below in Section A. β-Mannosidase activity was inthe heparin column flowthrough and was further purified as describedbelow in Section B.

A. β-Xylosidase.

The enzyme pool described above was dialyzed 2 hours against Buffer Abefore being applied to a column of Q-Sepharose (1.6×12 cm) equilibratedwith Buffer A. The column flowthrough which contained enzyme activitywas dialyzed 2 hours against Buffer C (20 mM potassium phosphate[pH6.0], 25 mM NaCl, 0.1 mM Na₂EDTA) before being applied to a column ofS-Sepharose (1.0×10 cm) equilibrated with Buffer C. The column waswashed with 20 ml Buffer C followed by a linear gradient of NaCl(0.025–0.95 M) in 150 ml of Buffer C (flow rate 1 ml/min; 2 mlfractions). The enzyme eluted between 0.4–0.55 M and pooled fractionswere dialyzed for 2 hours against Buffer A. After dialysis, the pool wasloaded onto a Heparin-TSK (3 ml) column equilibrated with Buffer A. Thecolumn was washed with 6 ml of Buffer A followed by a linear gradient ofNaCl (0.05–0.95 M) in 90 ml Buffer A (flow rate 1 ml/min; 1 mlfractions). The enzyme activity eluted between 0.2–0.3 M and the pooledfractions were dialyzed overnight in Buffer A. Sodium azide was added to0.02% before storing the enzyme at 4° C. A yield of 4,000 units ofsubstantially pure enzyme was obtained.

B. β-Mannosidase

(NH₄)₂SO₄ (66 g) was added to the column flow through (500 ml) to afinal concentration of 1 M (NH₄)₂SO₄ before being applied to a column ofPhenyl Sepharose (1.6×15 cm) equilibrated with Buffer B (20 mM Tris-HCl[pH7.5], 0.95M (NH₄)₂SO₄, 0.1 mM Na₂EDTA) The column was washed with 160ml of Buffer B followed by a linear decreasing gradient of (NH₄)₂SO₄(0.095–0.001 M) in 800 ml of Buffer B. The enzyme eluted between0.9–0.7M and pooled fractions were dialyzed 4 hours against Buffer C.The pooled enzyme was loaded onto a column of S-Sepharose (1.0×10 cm)equilibrated with Buffer C. The column was washed with 20 ml of Buffer Cfollowed by a linear gradient of NaCl (0.025–0.95 M) in 150 ml of BufferC (flow rate 1 ml/min, 2 ml fractions). The pooled enzyme was dialyzedfor 4 hours against Buffer D (20 mM Tris-HCl [pH7.5], 25 mM NaCl, 0.1 mMNa₂EDTA) before being applied to a Mono Q HR5/5 (1 ml) columnequilibrated with Buffer D. Activity was located in the column flowthrough 0.8 g of (NH₄)₂SO₄ was added to flowthrough before being appliedto a Phenyl Superose HR 10/10 (8 ml) column equilibrated with Buffer B.Activity was located in the column flowthrough. 0.8 g of (NH₄)₂SO₄ wasadded to a final concentration of 1.5 M before being re-applied to thePhenyl Superose HR 10/10 column equilibrated with Buffer E (20 mMTris-Hcl [pH7.5], 2.0 M (NH₄)₂SO₄, 0.1 mM Na₂EDTA). The column waswashed with 10 ml of Buffer E followed by a linear decreasing gradientof (NH₄)₂SO₄ (2.0–0.02 M) in 100 ml Buffer E (flow rate 1 ml/ min, 1.5ml fractions). The enzyme activity eluted between 1.0–0.85 M and pooledfractions were concentrated using a Centriprep concentrator (Amicon,Inc.—Beverly, Mass.) to 1 ml. The concentrated enzyme was dialyzedovernight against Buffer A. Sodium azide (0.2%) and BSA (0.1 mg/ml) wasadded before storing the enzyme at 4° C. A yield of 500 units ofsubstantially pure enzyme was obtained.

Example 10 Characterizing Glycosidases

A. β-Xylosidase

FIG. 16 shows the ability of β-Xylosidase isolated from Xanthomonasholcicola to cleave AMC-substrates 300: Manαl-6(Manαl-4) (Xylβ1-2)(Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc-Co (lanes 1-4) and 264:Xylβ1-4Xylβ1-4Xylβ1-4-Co (lanes 5 and 6). 5 units of β-Xylosidase wasadded to a 10 μl reaction mixture containing 1 nmol of AMC-substrate 300(lanes 3 and 4) in 50 mM sodium citrate pH6.0 supplemented with 5 mMCaCl₂, or 1 nmol of AMC-substrate 264 (lane 6) in 50 mM sodium citratepH4.5. 2 unites of α1-2,3 Mannosidase isolated from X. manihotis(Example 4) was included in some of the reactions (lanes 2 and 4) toexpose the β1-3 xylosyl linkage to hydrolysis by β-Xylosidase.Undigested substrates were included as controls (lanes 1 and 5). Markers(M) included were a disaccharide, 191: Galα1-3Gal-Co and atetrasaccharide, 202: Galα1-4GlcNACβ1-3Galβ1-4Reactions were incubatedat 37° C. for 2 hours before analysis by TLC as described in Example 2.As shown in FIG. 16, β-Xylosidase cleaved the β1-2 linkage ofAMC-substrate 300 (lane 3) only with α1-2,3 Mannosidase (lanes 2 and 3)was included in the reaction. When incubated without α-Mannosidase, nocleavage was observed (lane 4). β-Xylosidase also cleaved the β1-4linkages of AMC-substrate 264 (lane 6).

B. β-Mannosidase

FIG. 17 shows the ability of β-Mannosidase to cleave AMC-substrates 259:Man1-4Man1-4Man-Co (lanes 1 and 2) and 300:Manα1-6(Manα1-4)(Xylβ1-2)Manβ1-4GlcNAcβ1-4 (Fucα1-3) GlcNAc-Co (lanes3–8). 2.5 units of β-Mannosidase was added to a 10 μl reaction mixturecontaining 1 nmol of AMC-substrate 259 (lane 2) in 50 mM sodium citratepH5.5 or 1 nmol of AMC-substrate 300 (lanes 7 and 8) in 50 mM sodiumcitrate pH6.0 supplemented with 5 mM CaCl₂, 2 units of α1-2,3Mannosidase (lanes 4–7) isolated from X. manihotis (Example 4), 2 unitsof β-Xylosidase (lanes 5–7) isolated from X. holcicola (describedabove), 10 units or α1-6 Mannosidase (lanes 6 and 7) isolated from X.manihotis (Example 4) were included in some of the reactions to exposethe β1-4 mannosyl linkage to hydrolysis by β-Mannosidase. Undigestedsubstrates were included as controls (lanes 1 and 3). Markers (M)included a disaccharide, 191: Galα1-3Gal-Co and a tetrasaccharide, 202:Galβ1-4GlcNAcβ1-3Galβ1-4Glc-Co. Reactions were incubated at 30° C. for 2hours before analysis by TLC as described in Example 2. As shown in FIG.17, β-Mannosidase cleaved the β1-4 linkage of AMC-substrate 300 (lane 7)only when α1-2,3 Mannosidase, β-Xylosidase and α1-6 Mannosidase wereincluded in the reaction. When incubated without these enzymes, nocleavage was observed (lane 8). β-Mannosidase also cleaved the β1-4linkages of AMC-substrate 259 (lane 2).

Example 11 Method For Purification of β-mannosidase and β-xylosidaseFrom Xanthomonas oryzae

Fermentation of Xanthomonas oryzae.

Xanthomonas oryzae strain NEB 416 was grown in Difco Nutrient Broth. Thecells were incubated with aeration and agitation at 30° C. until latelogarithmic growth. The cells were harvested by centrifugation andstored frozen at −70° C.

Purification of β-mannosidase and β-xylosidase.

β-mannosidase and β-xylosidase were purified from 378 g of Xanthomonasoryzae cells obtained from a 100 1 fermentation. The cells wereresuspended in 1144 ml of Buffer A (20 mM Tris pH 7.5, 50 mM NaCl and 1mM EDTA) and lysed by passage through a Gaulin press. Cellular debriswas removed by centrifugation in a Sharples centrifuge. The supernatantwas passed over a DEAE-Sepharose FF column (5.0×16.0 cm) equilibratedwith Buffer A. The column was washed with 1500 ml Buffer A. Theflow-through and the first 500 ml of the wash from the DEAE column werecollected and loaded onto a Heparin Sepharose CL6B column (2.5×29.0 cm)equilibrated with Buffer A. The Heparin-Sepharose column was washed with750 ml Buffer A. For purification of the β-mannosidase 1203 g ofammonium sulfate was slowly added to the flow-through and the wash (2300ml) collected from the Heparin-Sepharose column, to bring theconcentration of ammonium sulfate up Lo 80% of saturation. Theprecipitation was incubated overnight at 4° C. with stirring. Theprecipitate was collected by centrifugation at 15,000×g for 20 min. Thepellet was resuspended in 350 ml Buffer A and dialyzed overnight inBuffer B (20 mM Tris pH 7.5 and 1 mM EDTA) containing 1 M ammoniumsulfate. After dialysis any precipitate remaining was removed bycentrifugation at 15,000×g for 20 min. So as not to overload the nextcolumn the supernatant (270 ml) was split into two sets which weretreated identically from this point on. 135 ml of the supernatant wasloaded onto A Phenyl-Sepharose 6 fast flow (low sub) column (2.5×29 cm)which had been equilibrated with Buffer B containing 1 M ammoniumsulfate. The Phenyl-Sepharose column was washed with 1000 ml Buffer Bcontaining 1 M ammonium sulfate. β-mannosidase was eluted from thecolumn with decreasing linear ammonium sulfate gradient from 1 M to 0.05M. 14 ml fractions were collected at a flow rate of 2 ml/min and assayedfor activity. The peak of activity eluting between 0.6 to 0.5 M ammoniumsulfate was pooled, dialyzed in Buffer C (20 mM KPO₄ pH 6.0, 0.1 mM EDTAand 10 mM NaCl) overnight and loaded onto a SP-Sepharose column (1.5×10cm) which had been equilibrated with Buffer C. The column was washedwith 80 ml Buffer C and the enzyme was eluted from the column with alinear NaCl gradient from 0.01 to 0.95 M. 3 ml fractions were collectedat a flow rate of 1 ml/min. The peak of activity eluting at 0.15 to 0.3M was pooled and dialyzed for 4 hr in Buffer B containing 1 M ammoniumsulfate. The dialyzed pool was loaded onto a Phenyl-Superose HR10/10column equilibrated with buffer B containing 1 M ammonium sulfate. Thecolumn was washed with Buffer B containing 1 M ammonium sulfate and theenzyme was eluted from the column with decreasing linear ammoniumsulfate gradient from 1 M to 0.05 M. 1.5 ml fractions were collected ata flow rate of 1 ml/min and assayed for activity. The peak of activityeluting between 0.7 to 0.6 M ammonium sulfate was pooled, dialyzed inBuffer A containing 0.02% sodium azide before storing the enzyme at 4°C. The yield of substantially pure mannosidase was 1.2×10³ units.

Purification of β-xylosidase from Xanthomonas oryzae was conductedsubstantially in accordance with the protocol described in Example 9.The yield was 500 units as determined in accordance with the methoddescribed in Example 7.

Characterizing the Purified β-mannosidase.

10 units of β-mannosidase purified as above from Xanthomonas oryzae wasfound to react with 0.5 nmol of substrate (Manβ1-4Manβ1-4Man-Co) in theabsence of cofactors in 50 mM Citrate Phosphate buffer pH 5.4 althoughthe enzyme was similarly active at pH in the range of 4.5 to 6.0.Incubation was carried out for 1 hr at 37° C.

Cloning of β-Mannosidase Gene

1. DNA purification: To prepare the DNA of Xanthomonas oryzae 1 g ofcell paste was resuspended in 3 ml 0.3M sucrose, 25 mM Tris (pH 8.0), 25mM EDTA and 2 mg/ml lysozyme. The suspension was incubated at 37° C. for10 min. Following the incubation 4 ml of 2×Kirby mix [2 g sodiumtri-isopropylnapthalene sulphonate, 12 g sodium 2-amino-salicylate, 5 ml2 M Tris-HCl, pH 8, 6 ml phenol (neutralized with Tris pH 8.0)] wasadded to the cell suspension and agitated for 1 min on a vortex mixer. 8ml of a 1:1 mixture of neutralized phenol and chloroform(phenol/chloroform) was added to the tube and the mixture was vortexedfor 15 sec. The cell lysate was subjected to centrifugation for 10 minat 12,000×g. The aqueous phase was transferred to a fresh tubecontaining 3 ml phenol/chloroform and agitated for 15 sec on a vortexmixer. The suspension was centrifuged for 10 min a 12,000×g. The upperphase was transferred to a fresh tube and 1/10 volume of 3 M sodiumacetate and an equal volume of isopropanol was added to the tube. Thecontents of the tube were mixed by inversion. The clot of DNA was hookedout of the tube with a sealed pasteur pipette and transferred to a tubecontaining 10 ml 70% ethanol. Once rinsed the DNA was dissolved in 5 mlof TE (10 mM Tris pH 8.0 and 1 mM EDTA), RNase was added to a finalconcentration of 40 mg/ml. The DNA was incubated for 30 min at 37° C.After incubation 1.5 ml of phenol/chloroform was mixed with the DNAsolution by vortexing for 15 sec. The suspension was centrifuged a12,000×g for 10 min. and the aqueous phase was removed to a fresh tube.A 1/10 vol. of 3 M sodium acetate and an equal volume of Isopropanol wasadded to the aqueous phase. The clot of DNA was hooked out of the tubewith a sealed pasteur pipette and transferred to a tube containing 10 ml70% ethanol. Once rinsed the DNA was dissolved in 2 ml of TE.2. Partial digestion: The purified DNA was cleaved with Sau3AI toachieve partial digestion as follows: 125 μl of DNA at 76 μg/ml in 10 mMBis Tris Propane-HCl pH 7.0, 10 mM MgCl₂, 100 mM NaCl, 1 mMdithiothreitol buffer with 100 μg/ml BSA was divided into one 400 μlaliquot and four, 200 μl aliquots. To the 400 μl tube was added 2 unitsof Sau3AI to achieve 1 unit of enzyme per 4.75 μg of DNA. 200 μl waswithdrawn from the first tube and transferred to the second tube toachieve 0.5 units Sau3AI/4.75 μg, and so on, each succeeding tubereceiving half of the previous amount of Sau3AI. The tubes wereincubated at 37° C. for 15 minutes heat-treated at 72° C. for 15 minutesthen subjected to electrophoresis in a 0.7% agarose gel inTris-Borate-EDTA buffer. DNA fragments ranging in size from about 9 to 2kb were collected by electrophoresing into DEAE anion exchange paper for2 hr. The paper was washed two times in 150 μl of a buffer containing0.1 M NaCl, 10 mM Tris pH 8.0. and 1 mM EDTA. Subsequently, the DNA waseluted from the paper by washing the paper four times with 75 μl of abuffer containing 1.0 M NaCl, 10 mM Tris pH 8.0 and 1 mM EDTA. Theresulting solution containing the DNA fragment was extracted with 300 μlphenol/chloroform followed by extraction with 300 μl chloroform andprecipitated with 1 ml absolute ethanol by placing in a dry ice/ethanolbath for 15 min. The DNA was pelleted at 14k rpm for 5 min. The pelletwas rinsed with 70% ethanol, air dried and resuspended in a final volumeof 10 μl 10 mM Tris pH 8, and 1 mM EDTA. The the purified fragments wereused as described in step 3 below.3 Ligation: The fragmented DNA was ligated to pUC19 as follows: 3 μg ofSau3AI-partially digested Xanthomonas oryzae DNA (10 μl) was mixed with1.5 μg of BamHI-cleaved and dephosphorylated pUC19 (1 μl). 4 μl of10×ligation mix (500 mM Tris pH 7.5, 100 mM MgCl₂, 100 mM DTT, 5 mM ATP)was added, plus 25 μl of sterile distilled water to bring the finalvolume to 39 μl. 1 μl of concentrated T4 DNA ligase (2×10⁶ U/ml) wasadded and the mixture was incubated at 37° C. for 2 hours. 10 μl of theligation was deionized by drop dialysis using a Millipore VS 0.025 μMfilter. The DNA was then electroporated into E. coli ED8767. The E. coliwas prepared for electroporation by growing up 1 l of cells to Klett50–80 in L-broth. The cells were chilled on ice for 15 to 30 min andthen pelleted in the cold at 4,000 rpm for 15 min. The pellet was washed2 times in ice cold sterile water and once in 10% glycerol. The washedpellet was resuspended in 1 to 2 ml of 10% glycerol to a final cellconcentration of 3×10¹⁰ cells per ml. The cells were frozen until neededin 100 μl aliquots at −70° C. To electroporate the DNA into the preparedcells, the cells were gently thawed and placed on ice. 40 μl of cellswere mixed with 10 μl of the ligated and dialyzed DNA. The mixture wasplaced into a cold 0.2 cm electroporation cuvette. A pulse ofelectricity at 12.5 kV/cm with a time constant of 4–5 msec was appliedto the DNA cell mixture. The E. coli was immediately diluted with 1 mlL-broth, allowed to grow at 37° C. for 30 min. and either plated on 150mm L-agar plates containing selective media. After overnight incubationat 37° C., clones expressing an β-mannosidase were screened as describedbelow.4. Screening for β-mannosidase clones. To screen for clones whichexpress β-mannosidase which is capable of cleaving a syntheticsubstrates such as para-nitrophenyl sugars, first the chromogenicsubstrate 4-methylumbelliferyl (4-MU)β-D-mannopyranoside was tried. Thissubstrate was added to 1.5% agar and used to overlay the selective platecontaining colonies. After overlaying, colonies producing activeβ-mannosidase can be identified by viewing the colonies with long waveultraviolet light (366 nm). In this experiment of the 2×10⁴ coloniesscreened using this substrate, no fluorescent colonies were isolated.Since no clones were isolated using this method an other method had tobe employed. To screen for clones expressing the β-mannosidase from X.oryzae a differential plating medium was used. The medium used is avariation on EMB agar which was used to screen for E. coli mutants whichare unable to utilize lactose as a carbon source (lac⁻). Traditional EMBagar contains lactose and two indicator dyes, eosin yellow and methyleneblue. When a strain of E. coli which is capable of fermenting lactose(lac⁺) grows on EMB agar the colonies appear dark purple to blackhowever, lac⁻ E. coli colonies are white due to their inability toferment lactose. To screen for clones expressing an β-mannosidase thelibrary (described above) was plated at a concentration of approximately30,000 cfu/ml on M9 minimal media containing 100 mg carbenicillin, 0.4 geosin yellow, 0.065 g methylene blue and 1 g α-mannobiose per liter ofmedium. The disacchride mannobiose (mannose β-1-4mannose) can not beutilized as a carbon source by E. coli unless they express the clonedβ-mannosidase which will cleave the mannobiose into mannose which can befermented by the host. The plates were incubated at 37° C. for 7 days.Two colonies out of 15,000 cfu plated were pigmented deep red. Thesecolonies were picked, streaked for isolated colonies on LB agarcontaining 100 ug/ml ampicillin and grown at 37° C. Isolated colonieswere picked into 5 ml LB with ampicillin and grown 6 hr at 37° C. Of the2 colonies tested, crude extracts from one showed β-mannosidaseactivity. Crude extracts prepared from isolated colonies which had beenpicked into LB with ampicillin and grown 18 hr at 37° C. showed noβ-mannosidase activity. This method has been used to isolate otherglycosidases (exo-α1-6 mannosidase and exo-α1-2,3-mannosidase fromXanthomonas manihotis) which do not cleave synthetic substrates or whichare unstable in E. coli. The only limitation are: 1) that the di-, tri-,or oligosacchride can not be utilized by the host unless theexo-glycosidase is present, 2) that the sugar released by theexo-glycosidase must be able to be utilized by E. coli as the solecarbon source and 3) that the sugar substrate must be available insufficient quantities that it can be added to the agar base to allowgrowth of the host expressing the exo-glycosidase.

Example 12 Method For Screening and Purification of an Exo-β1-4Galactosidase

Screening of Strains for β1-4 Galactosidase Activity

The method of screening is described in Example 2. The oligosaccharideGalβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3)Galβ1-4Glc-AMC was incubated withcell extracts at 37° C. for only 15 minutes followed by TLC analysis ofthe digestion products. An extract was selected only if at least bothnon reducing galactoses were removed. This criteria was established tobe able to select galactosidases that have a high specific activity forcomplex carbohydrates.

Fermentation of Bacteroides fragilis

Bacteroides fragilis strain 3392 (VPI Anaerobic Culture Collection) wasgrown in 1001 of TYG (Trypticase, Yeast Extract, Glucose Broth). Thecells were incubated anaerobically with minimal agitation at 37° C.until stationary phase growth. 416 g of cell paste was harvested bySharpels tubular centrifugation and stored frozen at −70° C.

Purification of the β1-4 Galactosidase from Bacteroides fragilis.

β1-4 Galactosidase was purified from 30 g of Bacteroides fragilis cellsremoved from the frozen cell paste of the 100 l fermentation. The cellswere resuspended in 100 ml of buffer A (20 mM Tris pH 7.5, 50 mM NaCland 0.1 mM EDTA) and lysed by sonication (30 second pulses at 50% dutycycle). Cellular debris was removed by centrifugation (15,300×g). Thesupernatant was passed over a DEAE-Sepharose FF column (1.6×15.0 cm)equilibrated with buffer A. The column was washed with 1500 ml buffer A.The flow-through and the first 90 ml of the wash from the DEAE columnwere collected and (NH₄)₂SO₄ was slowly added to final concentration of1.0 M. The pooled flow-through and wash was loaded onto a PhenylSepharose FF column (1.5×45 cm, 80 ml) which had been equilibrated withbuffer B (20 mM Tris pH 7.5, 1.0 M (NH₄)₂SO₄, 50 mM NaCl and 0.1 mMEDTA) . After loading, the Phenyl-Sepharose column was washed with 240ml buffer B. β1-4 Galactosidase was eluted from the column withdecreasing linear ammonium sulfate gradient from 1 M to 25 mM. 10 mlfractions were collected at a flow rate of 2 ml/min and assayed foractivity as described in the Example 2. The branched oligosaccharideGalβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3) Galβ1-4Glc-AMC was used as asubstrate since this substrate is not cut by an endo-β-galactosidasepreviously reported. The peak of activity eluted between 0.7 to 0.6 M(NH₄)₂SO₄ was pooled, dialyzed in buffer A overnight and loaded onto aHeparin-Sepharose FF column (1.6×10 cm) which had been equilibrated withbuffer A. The column was washed with 60 ml buffer A and the enzyme waseluted from the column with a linear NaCl gradient from 0.01 to 0.95 M.3 ml fractions were collected at a flow rate of 2 ml/min. The peakactivity eluted at 0.25 to 0.40 M NaCl. The pooled activity was dialyzedfor 4 hours in Buffer A. The dialyzed pool was loaded onto a Mono QHR10/10 prewashed with buffer A. The peak activity eluting in the flowthrough and 30 ml wash with buffer A was pooled and loaded onto aHeparin TSK ( 0.7×7.5 ml; 3 ml) which had been equilibrated with bufferA. The column was washed with 9 ml Buffer A followed by a 60 ml NaClgradient from 0.50 M to 0.950M. 1.5 ml fractions were collected at aflow rate of 1 ml/min. The peak of activity eluting between 0.35 to 0.4M NaCl was pooled. The yield of substantially pure β1-4 Galactosidasewas 2.0×10³ units. One unit of enzyme was defined as the amount ofenzyme required to release both terminal galactoses from 1 nmole of theoligosaccharide substrate:

Galβ1-4GlcNAcβ1-6(Galβ1-4GlcNAcβ1-3)Galβ1-4Glc-AMC at 37° C. in 1 hour.

Characterizing the Purified Exo-β1-4 Galactosidase.

1.0 units of β-galactosidase purified as above from Bacteroides fragiliswas found to react with 0.5 nmol of substrateGalβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-4Glc-AMC in 50 mM Citrate buffer pH 6.0although the enzyme was similarly active at pH in the range of 4.5 to6.0. Incubation was carried out for 1 hr at 37° C. No cofactors werefound to be required for activity. The purified enzyme will also cleavethe following substrates:

-   p-Nitrophenyl-β-Gal Galβ1-4Clc-AMC-   Galβ1-4GlcNAcβ1-6(Galβ1-3GlcNAcβ1-3)Gaβ1-4Glc-AMC (1-4linkage only)-   Galβ1-4GlcNAc:1-6(Galβ1-4GlcNAcβ1-3)Galβ1-4Glc-AMC-   Galβ1-4GlcNAcβ1-2Manβ1-6Manβ1-4GlcNAc-AMC-   Galβ1-4GlcNacβ1-4Manα1-3[Galβ1-4GlcNAcβ1-2(Galβ1-4GlcNA-   cβ1-2)Manβ1-6]Manβ1-4GlcNAcβ1-4GlcNAc-AMC-   Galβ1-4(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc-AMC only after removal of    Fucose first    The purified enzyme does not cut the following Gall-2,3,6 linkages    in the following substrates:-   Galβ1-4GlcNAcβ1-6(Galβ1-3GlcNAcβ1-3)Galβ1-4GlcNAcβ1-4Glc-AMC-   Galβ1-3GlcNAcβ1-4GlcNAcβ1-4Glc-AMC-   Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-4Glc-AMC-   Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4GlcNAcβ1-4Glc-AMC    (with or without removal of fucoses)-   Glaβ1-2 (Arabβ1,3)GlcNAc-AMC-   Xylα1-6(Galβ1-2Xylα1-6Glcβ1-4) (Galβ1-2Xylα1-6Glcβ1-4) Glc-AMC-   Galβ1-6GlcNAc

1. A method for cleaving a glycosidic bond in a carbohydrate, comprisingthe steps of: (a) adding to the carbohydrate, a glycosidase of definedsubstrate specificity obtainable from a Xanthomonas species wherein theglycosidase is selected from a β1-3>>4 galactosidase, an α1-2,3mannosidase, a β-glucosidase, an α1-3,4 fucosidase, an α1-2 fucosidase,a β-N-acetylglucosaminidase, β-N-Acetylglucosaminidase, an α1-6mannosidase, an α1-3,6 galactosidase, an α1-3,6 mannosidase, aβ-xylosidase and a β-mannosidase; and (b) cleaving the glycosidic bondbetween constituent monosaccharides of the carbohydrate by means of theglycosidase.
 2. A method according to claim 1, wherein the cleavedcarbohydrate has altered immunogenic properties compared with thecarbohydrate prior to cleavage.
 3. A method according to claim 1,wherein the defined substrate specificity is determined using afluorescent chromophore.
 4. A method according to claim 3, wherein thefluorescent chromophore is 7-aminocoumarin.
 5. A method according toclaim 1, further comprising: determining cleavage of the glycosidic bondby thin layer silica gel chromatography.